Soil waterlogging has become a major factor affecting the growth, development and survival of many plant species, not only in natural ecosystems, but also in agricultural and horticultural systems (Dat et al., 2006). Transient flooding periods are frequently observed as a result of over-irrigation, inadequate drainage, the removal of vegetation cover and/or global warming. In addition, climate predictions suggest that the occurrence of this event will increase in frequency in the near future. Some of the best characterized plant adaptations to hypoxia include a switch in biochemical and metabolic processes commonly observed when O2 availability becomes limiting (Dat et al., 2004). Most plant species synthesize a set of c. 20 anaerobic proteins (ANP) that enable an oxygen-independent energy-generating metabolism to proceed when conditions become unfavourable for aerobic energy production (Subbaiah & Sachs, 2003). Other observed adaptations include the formation of hypertrophied lenticels, the development of aerenchyma, root cortical air spaces that enhance the efficiency of gas transfer between aerial and submerged organs, as well as the promotion of adventitious roots (Vartapetian & Jackson, 1997; Jackson & Colmer, 2005; Folzer et al., 2006).
In contrast to the wealth of data available concerning the molecular and cellular mechanisms of hypoxia sensing and signalling in animals, such mechanisms have been more rarely described in plants, even less in woody species. In mammals, the hypoxia-inducible heterodimeric transcription factor (HIF) is a key regulatory element in the response to hypoxia (Giaccia et al., 2004; Semenza, 2004). However, to date no such sensor has been identified in plants (Bailey-Serres & Chang, 2005; Agarwal & Grover, 2006). Recent broad-range approaches (DNA chip technology or proteome analysis) have helped to identify novel genes and proteins involved in plant responses to soil waterlogging and anaerobiosis (Chang et al., 2000; Klok et al., 2002; Agarwal & Grover, 2005; Branco-Price et al., 2005; Liu et al., 2005; Loreti et al., 2005). However, novel components of the signal transduction pathway leading to hypoxia-induced gene expression have been documented exclusively in crop plants (Lasanthi-Kudahettige et al., 2007) and model organisms such as Arabidopsis thaliana (Miyashita et al., 2007). These components include rapid changes in cytosolic Ca2+ levels (Snedden & Fromm, 1998; Subbaiah et al., 1998, 2000; Luan et al., 2002) and Ca2+-binding proteins (Folzer et al., 2005), the induction of ethylene biosynthesis (Drew et al., 2000; Nie et al., 2002), Rop (RHO-related GTPase of plants) G-protein signalling (Baxter-Burrell et al., 2002), as well as a large number of transcription factor families (AtMYB2, ZAT 12, WRKY factors; Bailey-Serres & Chang, 2005). In contrast to the amount of data available for herbaceous species, very little is known about the molecular mechanisms that underlie the sensing and signalling to hypoxia in woody species. This is especially apparent with forest tree species, which are not only of primary interest to the wood industry, but are also critical for the preservation and/or conservation of forest biodiversity.
Recently, additional insight into the response of plants to hypoxia has been provided by the discovery of stress-induced genes that affect plant metabolism and growth under low oxygen tensions (Dordas et al., 2003a). Among these, hemoglobins (Hbs) are ubiquitous molecules that have been found in various species from most of the taxonomic kingdoms, including bacteria, yeasts, protists, plants and animals (Wittenberg & Wittenberg, 1990; Hardison, 1996; Suzuki & Imai, 1998). All Hbs contain a heme group carrying an iron ion, which is responsible for the reversible binding to gaseous ligands such as oxygen (O2) and carbon monoxide (CO) (Weber & Vinogradov, 2001). In plants, at least three different Hb families have been identified: symbiotic, nonsymbiotic and truncated Hbs (Ross et al., 2002). Symbiotic Hbs, or leghemoglobins, are specifically synthesized in nitrogen-fixing legume root nodules, and their main function is to facilitate oxygen transport and scavenging to protect Rhizobium nitrogenase from inactivation (Appleby, 1984). Plant truncated Hbs are short versions of the classical globin fold. The function of these proteins, recently detected in organs of angiosperm species such as Arabidopsis (Watts et al., 2001) and wheat (Larsen, 2003), is still unknown. Finally, nonsymbiotic Hbs occur at much lower abundance, but appear ubiquitous in all plant species examined (Dordas et al., 2003a). In vascular plants, two classes occur. Class 2 nonsymbiotic Hbs present similar O2-binding properties to those of symbiotic Hbs and are inducible by cold stress (Trevaskis et al., 1997) or cytokinin treatment (Hunt et al., 2001). In contrast, class 1 nonsymbiotic Hbs have high O2 affinity and are induced under hypoxic conditions (Duff et al., 1997; Trevaskis et al., 1997). Because of an extremely low O2-dissociation constant, class 1 nonsymbiotic Hbs might not function as O2 carriers, as originally thought. In fact, recent studies suggest that their presence could regulate cellular nitric oxide (NO) levels, thus improving the redox and/or energy status of the plant cell during hypoxia (Dordas et al., 2003b; Perazzolli et al., 2004).
In an attempt to gain further understanding of the difference in the molecular responses of tree species to hypoxia, the cloning and characterization of a nonsymbiotic Hb gene from sessile oak was undertaken. The analysis was further complemented by comparing the expression profile of the gene in two oak species with a contrasted response to flooding. The genus Quercus (oaks), which includes over 300 woody species, is widespread in the northern hemisphere, where it represents the dominant vegetation of temperate forests (Nixon, 1993). We decided to focus on the two predominant European oak species, pedunculate and sessile oak, to investigate the spatial and temporal expression patterns of Hb during the early response to hypoxia. The two species generally cohabit in forest ecosystems; however, sessile oak is found more frequently on well drained soils, whereas pedunculate oak can populate poorly drained sites where temporary waterlogging occurs (Lévy et al., 1992).