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Heterophylly in Aquatic Plants
Institute of Molecular Biology, Academia Sinica, Taiwan, Republic of China and Molecular and Cell Biology Division, Development Center for Biotechnology, Taiwan, Republic of China
Many aquatic plants produce distinct types of leaves in response to changes in natural habitat and the cultural conditions in the laboratory. When conditions are in favor of producing leaves of the submerged type, application of the plant hormone abscisic acid (ABA) induces formation of aerial type morphology. The leaf characteristics induced by ABA are indicative of adaptation to drought and photosynthetic parameters in the aerial environment. The ABA effect is seen in various aspects of leaf determination, and each tissue has a specific response. Similar to nature, heterophyllous switch induced by ABA is reversible. A window of responsiveness exists in target tissues localized at the shoot apex where a number of regulatory and metabolic genes respond early during ABA induction. Studies of heterophylly point to a role of ABA in regulating developmental plasticity and in the crosstalk of the signals for photosynthesis and photomorphogenesis.
Abscisic acid is well known for its involvement in reproduction and stress response, and is often regarded as a negative regulator due to the induction of dormancy in seed and bud, and the inhibition of seed germination and vegetative growth in many experimental systems (Zeevaart and Creelman 1988; Leung and Giraudat 1998). However, in a number of aquatic plants, exogenous application of ABA triggers heterophyllous switch during the adult vegetative phase, which, in most tissues, results in the stimulation of growth and development. In those plants, ABA induces the formation of aerial type morphology that is distinct from its counterpart submersed in water, particularly in the size and shape of the leaf. This ABA effect has been seen in phylogenetically diverged taxa, including a fern (Marsilea, Liu 1984), a monocot (Potamogeton, Anderson 1978), and several dicots (Limnophila, Mohan Ram and Rao 1982; Callitriche, Deschamp and Cooke 1984; Ranunculus, Young and Horton 1985; Hippuris, Kane and Albert 1987a; Proserpinaca, Kane and Albert 1987b). Like other physiological functions, ABA appears to integrate the environmental and developmental cues and to turn on and off a developmental program.
Heterophylly in aquatic plants is reversible and depends on the environment. In nature, heterophyllous transition occurs in response to changes in water level and the seasons (Allsopp 1965; Sculthorpe 1967). In general, leaves produced in submerged shoots are dissected or linear, with few stomata, no cuticle, and undifferentiated mesophyll; whereas aerial leaves and those floating on the surface of water are entire and broad with stomata and epidermal cuticle, and the mesophylls differentiating into spongy and palisade tissues (see Zeevaart and Creelman 1988; Lin and Yang 1999 and the literature cited therein). The submerged, floating, and aerial leaves are all adult forms that are alternately produced during the year. Normally, the submerged type leaves are produced in the winter, the floating or aerial type leaves are produced in the summer, and intermediate type leaves may be produced during the transitions (Allsopp 1965; Sculthorpe 1967).
There is apparent interplay among environmental factors (McCully and Dale 1961; Bostrack and Millington 1962; Wallenstein and Albert 1963; Allsopp 1965; Gaudet 1965; Schmidt and Millington 1968; Cook 1969; Bodkin et al. 1980; Maberly and Spence 1989; Trewavas and Jones 1991; Titus and Sullivan 2001). In summer months, aerial type leaves are produced even though the shoots are immersed in water, sometimes as deep as one and a half meters below the water surface. This phenomenon questions what factors in water level changes and seasons are the major triggers for developmental switch? Extensive physiological studies in various plant species show that, each environmental factor, at an optimal range, is capable of inducing the switch from developing one type of leaf to another (for reviews, see Allsopp 1965; Maberly and Spence 1989; Trewavas and Jones 1991). Generally, high temperature, long photoperiod, high light intensity, blue light, far-red light, osmotic stress, and low CO2 partial pressure favor the formation of aerial type morphology. These factors interact with each other, and the effects are both qualitative and quantitative. Ambiguous results and conflicting data in the literature are often due to variations in the control settings of the experiment. Thus, heterophylly may be seen as an adaptive mechanism that is sensitive to all environmental parameters and that responds accordingly to maximize the capability for survival.
Of course the next question would be: Since ABA mimics changes in the environment, is it the endogenous factor mediating the environmental changes? If so, how are the changes perceived and how do they manipulate ABA content? What is the physiological relevance of the morphology induced by ABA? And, no matter whether ABA is the endogenous factor, how does ABA signal transduction connect to the developmental programs and trigger a switch? New leaf formation requires an orchestration of specifically oriented cell division, expansion, and differentiation. How does ABA affect these processes? What are the primary targets of ABA in the morphological determination and what are the secondary effects? Is there a "master switch" (or "master switches") for heterophylly? Although heterophyllous induction seems unique to aquatic plants, these questions are fundamental to ABA hormone biology. Many questions currently exist for the fascinating manifestation of developmental plasticity in the adult vegetative phase. Through the studies in various systems, some of the questions can now be addressed.
ABA Mediates Some But Not All Environmental Signals
Like other responses to water stress, ABA mediates the signals of water and nutrition status that induce heterophyllous switch. Both in Hippuris vulgaris and Marsilea quadrifolia, the increase in endogenous ABA level correlates with morphological changes in a modified culture medium during a time course (Goliber and Feldman 1989; Lin and Yang 1999). In H. vulgaris, ABA content also increases in response to an inducing dose of photon fluence and far-red light (Goliber 1989), suggesting ABA involvement in light intensity and phytochrome signaling. However, in M. quadrifolia (Figure 1) blue light induction of heterophylly does not require de novo synthesis of ABA nor does it cause accumulation of ABA beyond the control level (Lin and Yang 1999). Therefore, independent signaling pathways exist to regulate heterophyllous determination, and some but not all are mediated by ABA (Lin and Yang 1999).
ABA effect on the formation of aerial characteristics is dose-dependent and usually starts at the submicromolar level within the physiological range (Anderson 1982, Mohan Ram and Rao 1982; Liu 1984; Kane and Albert 1987a,b). In M. quadrifolia, ABA content in aerial leaves of plants grown in a greenhouse or in the field is similar to the contents of that in submerged leaves produced in aseptic culture (Lin and Yang 1999); aerial morphology does not necessarily correlate with elevated ABA levels. One possibility to be examined is the environmental factor blue light. Blue light may cause redistribution without increasing the overall content of ABA; the amount of ABA in the responsive target tissues may reach beyond a threshold to trigger the heterophyllous switch.
ABA and Adaptation: Drought, Photosynthesis, and Photomorphogenesis
In aerial leaves, cuticle formation and stoma differentiation are indications of drought adaptation, whereas the broader, less dissected lamina and the differentiation of mesophylls into palisade and spongy tissues, suggest a photosynthetic relevance. In Ranunculus flabellaris, the aerial leaves have higher chlorophyll content and more chloroplasts than the submerged leaves (Young et al. 1987; Young et al. 1990). Measurements of photosynthetic rates and other related parameters show that in their respective environment, the aerial and the submerged leaves have similar photosynthetic capacities, yet the overall photosynthetic output of the predominant morphology in the given environment is higher (Maberly and Spence 1989; Nielsen and Sand-Jensen 1993). Therefore, each type of morphology is well adapted in terms of both drought and photosynthesis.
Because ABA induces formation of aerial leaves in submerged shoots, the messages for drought and photosynthesis appear to be coupled and mediated by ABA. Further, in H. vulgaris the change in ABA level correlates with high light induction and phytochrome suppression of aerial morphology (Goliber 1989). Thus, ABA seems to mediate the signal for photomorphogenesis as well. However, by treating M. quadrifolia simultaneously with fluridone and blue light, aerial leaves can be induced without ABA biosynthesis (Lin and Yang 1999, see Figure 1). In those plants, the synthesis of chlorophylls and carotenoids are also inhibited, hence the capability of photosynthesis to be separate from photomorphogenesis (Lin and Yang 1999). Taken together, the results confirm the role of ABA in the adaptation to drought, the photosynthetic parameters corresponding to the aerial environment, and the crosstalk of the signals for photosynthesis and photomorphogenesis.
Leaf Determination Is Progressive: Continuous Presence of ABA Is Required for Completing the Morphogenetic Transition
Similar to leaf development, heterophyllous determination in aquatic plants is a graded process (Deschamp and Cooke 1984; Goliber and Feldman 1990; Young et al. 1995; Bruni et al. 1996; Gee and Anderson 1998; Lin and Yang 1999). A distinct ABA effect is seen in each step up to very late stages, including the determination of size, shape, and dissection pattern of the lamina, the differentiation of mesophyll, aerenchyma, and stomata, and the formation of epicuticular layer. Addition or removal of exogenous ABA often results in a gradient of intermediate morphology, depending on the concentration used and the timing of application. The full complement of aerial characteristics is produced only when ABA is continuously present throughout the developmental course of a leaf (Goliber and Feldman 1990; Young et al. 1995; Hsu et al. 2001). These data suggest that ABA action in heterophylly is transduced through a ligand-receptor type signaling circuitry, rather than a once-for-all type switch.
Target Tissues of ABA Are Located at the Shoot Apex, Each Tissue Having a Distinct Morphogenetic Response
ABA effect is seen in leaves that are developing and newly emerged or initiated, those fully grown prior to ABA treatment are unaffected (Young et al. 1995; Gee and Anderson 1998; Hsu et al. 2001; Figure 2). In M. quadrifolia, all three organs of the vegetative phase (root, stem, and leaf) respond to ABA (Liu 1984; Hsu et al. 2001; see Figure 2). The root system is adventitious and derived from the shoot apical meristem. The changes in root morphology and the elongation and branching, are dramatic and are an integral part of the ABA-induced morphogenesis (Liu 1984; Hsu et al. 2001; see Figure 2). These observations indicate that target tissues of ABA are located at the shoot apices, and each tissue has a distinct response that incorporates into a genetic program encoding the aerial morphology (Hsu et al. 2001). In accord with the hypothesis that a ligand-receptor type circuitry governs ABA signaling, it is reasonable to suggest that individual tissues respond to ABA through specific paths that lead to unique pattern of development. It does not exclude the possibility that ABA signal transduction acts on "master switches" that dictate a hierarchy of controls regulating the sequential temporal and spatial morphogenetic events.
A Number of Regulatory and Metabolic Genes Respond Early to ABA
In search of the molecular basis of heterophyllous switch, a number of ABA regulated early genes are identified from the shoot apex tissues of M. quadrifolia (Hsu et al. 2001). These genes are designated ABRH for ABA-responsive heterophylly. Transcript levels of ABRH change within half an hour to an hour following the addition of ABA to the culture medium. Some changes are transient while others are persistent. Based on sequence homology, many genes transiently regulated by ABA appear to have a regulatory role, encoding transcription factors, membrane transporters, and protein kinases. Several persistently regulated genes show homology to genes involved in aspects of cell growth, such as plastid biogenesis. Of the 24 genes identified, seven are primary response genes. It is noted that genes encoding transcription factors, in particular, leucine zipper type proteins (known to regulate ABA response), are present among the primary and secondary genes, suggesting the possibility of a hierarchy of transcriptional regulation. Functional analysis of these ABRH genes will be an important step toward unraveling the molecular mechanism involved in ABA induction of heterophyllous switch.
Heterophyllous control in aquatic plants is complex and intriguing. It provides a system for studying the fundamental mechanism of developmental plasticity, hormonal signaling, the interaction between environmental and endogenous factors, and the crosstalk among signaling pathways. Through successive efforts of researchers, the role of ABA is revealed—to mediate the environmental signals for the adaptation to drought and to maximize photosynthetic capacity. It also orchestrates the morphogenetic events that fulfill the requirement for such adaptation. Although the central questions of the molecular mechanisms for heterophylly remain unanswered, with bountiful physiological data in the literature, an array of ABA responsive early genes identified in the shoot apex target tissues, and the advance of functional genomics tools, an integrated picture is now in sight.
Research in my lab on heterophylly has been supported by grants from Academia Sinica and National Science Council, Republic of China (NSC89-2311-B-001-134; NSC90-2311-B-169-001; NSC90-2311-B-169-002).
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