Plant Biotechnology 25, 257–263 (2008)

Review

Wild plant resources for studying molecular mechanisms of drought/strong light stress tolerance

Kinya Akashi1,*, Kazuya Yoshimura2, Yoshihiko Nanasato3, Kentaro Takahara4, Yuri Munekage1, Akiho Yokota1 1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan; 2 Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University, Aichi 487-8501, Japan; 3 Research Center for Green Science, Fukuyama University, Hiroshima 729-0292, Japan; 4 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan * E-mail: [email protected] Tel: 81-743-72-5563 Fax: 81-743-72-5569

Received February 13, 2008; accepted March 25, 2008 (Edited by K. Yazaki)

Abstract Drought is one of the major environmental factors restricting plant productivity worldwide. Under drought in the presence of strong light, plants are liable to be damaged by excessively-absorbed solar energy as well as dehydration of their tissues. Wild plants found in the arid zones are equipped with specialized mechanisms for either avoiding or tolerating drought, and successfully adapt to the harsh environments. Recent molecular studies on the wild plants have shed new lights on the unique features of their resistance mechanisms, which are markedly different from those found in the model and/or domesticated crop plants. Key words: Wild plant, drought/strong light tolerance.

In nature, plants are exposed to various adverse exposed to multiple physicochemical constraints under environmental conditions, which restrict their drought/high light stresses, ranging from excess light productivity and even their survival. Water deficits are energy, dehydration and high temperature. estimated to be the prime factor suppressing net primary To adapt to the drought and strong light environments, production of the vegetation of the earth (Larcher 1995). terrestrial plants has developed various mechanisms in This suppression is predominant in the arid and semi- evolution. These adaptive mechanisms include those for arid zones between the latitudes 15° and 30°N and S, stress-avoidance as well as stress-tolerant strategies, and which occupies ca. 40% of the earth’s land. Even in the are particularly manifest in wild plants inhabiting arid temperate zone, water shortage is recognized as a prime and semi-arid regions. In this mini-review, we would like factor limiting crop yield (Boyer 1982). to focus on recent physiological, biochemical and Water is an absolute requirement for plant growth and molecular studies on these plants, in which diverse development. In C3 plants, the amount of water lost by strategies for coping with drought/strong light stresses leaf transpiration through stomata is estimated to be 500 have been elucidated. to 1000 times larger than that of CO2 assimilated in the leaves on a molar basis (Larcher 1995; Yokota et al. 2006). Therefore, assimilation of carbon via Xerophytes as model systems for studying photosynthesis incurs huge water costs in plants. drought/strong light stress tolerance Moreover, water transpiration from the leaves is Plants which survive in the environments with limited important for lowering the leaf temperature (Nobel water resources are called xerophytes (xero means dry, 1999). As soil water content decreases under drought in phyte for plant), which include at least 20,000 the presence of strong solar radiation, plants close their economically-useful plants (Wickens 1998) and possess stomata, and photosynthetic CO2 fixation is strongly a number of mechanisms to adapt to dry habitats. suppressed (Cornic 2000). Under such conditions, Survival strategies employed by these plants are excessively absorbed light energy that cannot be categorized into three groups (Larcher 1995); (i) consumed through photosynthetic metabolism is liable to drought-escaping strategy is observed typically in bring an increased risk of oxidative injury and ephemeral plants, which germinate and complete their photoinhibition (Price et al. 1989). Therefore, plants are cycle of vegetative growth when water is available in the

This article can be found at http://www.jspcmb.jp/ 258 Wild plant resources for drought tolerance

short rainy season. Most plants of this kind survive dry in the leaf cells (Bianchi et al. 1991). Water deficits seasons as inert, desiccation-tolerant forms such as seeds induce conversion of 2-octulose to sucrose, which or dormant organs. (ii) Desiccation-avoidance strategy accumulates in large quantity in the desiccated leaves. involves maintenance of the hydration status of plant Accumulation of sucrose has been commonly observed tissues despite dryness of the surrounding air and soils. in many resurrection plants, which may confer (iii) Desiccation-tolerant strategy represents the ability desiccation tolerance by inducing vitrification of the for tolerating severe desiccation of the plant tissues. cytosol, thereby stabilizing the cellular macromolecules Typical examples employing this strategy are observed in and intracellular structure under desiccation (Ingram and so called “resurrection plants”, which are characterized Bartels 1996). Molecular studies revealed that global by the ability to tolerate nearly complete desiccation of change in the gene expression patterns in the early phase their tissues, and by the surprisingly rapid recovery from of water deficits in the leaves of C. plantagineum their desiccation state to the photosynthesis-competent (Ingram and Bartels 1996; Bartels and Salamini rehydration state upon resuming water supply (Gaff 2001). These stress-inducible factors include late 1977). For example, Craterostigma plantagineum (Figure embryogenesis-abundant (LEA) proteins, enzymes for 1A) from South Africa is tolerant to severe desiccation in sugar metabolism, transporters for sugars, and factors for which 85% of the initial fresh weight of the leaves is lost, transcriptional regulation and signal transduction, and its photosynthesis and other metabolisms are rapidly suggesting the occurrence of complex interplay between recovered to the original levels upon resuming irrigation these factors for establishing dehydration tolerance in (Bartels et al. 1990; Bartels and Salamini 2001). this plant. Biochemical and molecular studies on C. An experimental system for genetic transformation is plantagineum have offered interesting insights on how available in C. plantagineum, which enabled to isolate these resurrection plants survive extreme desiccation. regulatory factors involved in the desiccation tolerance in Under the rehydration state, C. plantagineum this plant. The CDT-1 gene isolated by activation tagging

accumulates unusual C8 sugar 2-octulose up to 400 mg/g was shown to be essential for dehydration tolerance in dry weight, which constitutes 90% of the soluble sugars this plant (Furini et al. 1997). CDT-1 encodes a

Figure 1. Wild plants with drought/strong light stress tolerance. (A) C. plantagineum (resurrection plant; adapted from Bartels et al. 1990). (B) N. gulauca (tree tobacco; Cameron et al. 2006). (C) M. crystallinum (ice plant; Adams et al. 1998). (D) P. euphratica (Brosche et al. 2005). (E) C. lanatus (wild watermelon). (F) Retama raetam (Mittler et al. 2001). Reprinted with permissions of Planta (A), Plant Physiology (B), New Phytologist (C) and Plant Journal (F). K. Akashi et al. 259 regulatory RNA or short peptide involved in the reduction cycle (Dodd et al. 2002). Water-use-efficiency activation of ABA signal transduction pathway. CDT-2 in CAM plants is generally 3 to 10 times higher than gene isolated by a similar screening procedure showed those in C3 plants (Larcher 1995). high sequence similarity to CDT-1, suggesting that this CAM occurs in approximately 6% of the terrestrial CDT1/2 family play an important role in the signal angiosperms, and displays enormous species-specific transduction for desiccation tolerance in this plant variations concerning the metabolic scheme and its (Smith-Espinoza et al. 2005). No homologous genes regulation (Dodd et al. 2002). One example is illustrated have been found in the Arabidopsis genome, suggesting by the facultative CAM species, which operate C3 that this plant develops unique signal transduction photosynthesis at times of sufficient water supply but system to adapt to the severe environmental conditions. switch to CAM during periods of water limitation (Cushman and Borland 2002). In Mesembryanthemum crystallinum (Figure 1C), induction of CAM is Mechanisms for maintaining water status accompanied with the up-regulation of the key enzymes Minimizing loss of water from the plant tissues offers an such as phosphoenolpyruvate carboxylase and NADP- obvious advantage to xerophytes, especially for the ones malic enzyme (Adams et al. 1998; Cushman and Bohnert employing desiccation-avoidance strategy. Stomata in 1999; Cushman and Borland 2002). Pharmacological this kind of xerophytes are typically smaller in size than studies have indicated that intracellular Ca2 signal is those found in mesophytes, and often hidden in the implicated in the induction of these genes (Taybi and grooves on the lower side of a leaf with dense coverage Cushman 1999). A gene for drought-induced Ca2- of hairs, which effectively reduce water transpiration dependent protein kinase McCPK1 was characterized in under dry air (Larcher 1995). QTL analyses in M. crystallinum, which undergoes stress-induced Arabidopsis also suggested importance of stomatal reversible change in its subcellular localization from the morphogenesis in water-use-efficiency of plants (Masle plasma membranes to nucleus and endoplasmic et al. 2005), which revealed that ERECTA, a gene for reticulum, suggesting the involvement of this protein in leucine-rich repeat receptor-like kinase, was a major the Ca2 signal transduction under stress (Chehab et al. determinant for water-use-efficiency of this plant via 2004). To understand molecular basis of CAM induction affecting stomatal density and epidermal cell process, integrative genomic approaches including large- morphogenesis. scale EST analysis (Kore-eda et al. 2004) and DNA Thick cuticle layers are advantageous for suppressing microarray (Cushman and Borland 2002) have been water loss from the leaves (Nawrath 2006). In a tree conducted in this plant. tobacco Nicotiana gulauca (Figure 1B) which inhabit in Water storage as well as efficient water acquisition are the arid region of South America, drought-induced the important traits for plants in the arid and semi-arid deposition of cuticular wax was temporally correlated zones. Baobab (Adansonia rubrostipa) stores huge with the up-regulation of a gene for lipid transfer protein amount of water in the stem, and prepares for the long- (Cameron et al. 2006), suggesting that the fortification of term water deficits (Chapotin et al. 2006). Succulent cuticle layer under stress is genetically controlled in this plants such as Opuntia acanthocarpa and Echinocereus wild plant. Genetic studies in Arabidopsis and Z. mays engelmannii accumulate massive amount of water- have led to the identification of many genes involved in binding mucilage in their stem up to 35% of their dry the biosynthesis of cutin and wax compounds (Nawrath weight, which serves as a large capacitor for water 2006). For example, shine (shn) gain-of-function mutant (Nobel 1992). Populus euphratica (Figure 1D) is a isolated by activation-tagging was characterized by the drought-tolerant woody species found in Central Asia increase in wax deposition in the cuticle layer, which and Middle East, which possesses developed root system conferred increased tolerance to drought in this mutant for the access to deep water tables (Hukin et al. 2005). compared to wild type (Aharoni et al. 2004). The SHN Comprehensive analyses including large-scale EST, gene encoded an AP2/EREBP-type transcriptional DNA microarray and metabolome have been reported in factor, which regulates wax biosynthetic genes. this plant (Brosche et al. 2005).

Crassulacean acid metabolism (CAM) is known as Wild watermelon (Citrullus lanatus; Figure 1E) is a C3 one of the adaptive mechanisms for performing plant inhabiting in the Kalahari Desert in Africa (Yokota photosynthesis under water-limited environments et al. 2002). Physiological study has shown that root (Ehleringer and Monson 1993). In CAM plants, stomata development of this plant was significantly stimulated in open at night, and atmospheric CO2 is initially fixed as a the early phase of drought stress (Yoshimura et al. 2008). C4 acid, which is then stored in the vacuole of the Proteome analysis in the root has revealed that drought photosynthetic cells. In the daytime, CO2 is released by stress induced a set of proteins related to root decarboxylation of the C4 acid, which is then re-fixed by morphogenesis and carbon/nitrogen metabolisms in the the operation of RuBisCO in the photosynthetic carbon early phase of the stress, whereas factors related to lignin 260 Wild plant resources for drought tolerance

biosynthesis and molecular chaperones are up-regulated monodehydroascorbate and H2O (Farver et al. 1994), in the later phase (Yoshimura et al. 2008). These accumulated abundantly in wild watermelon leaves in observations suggested that this xerophyte switches comparison to that in the domesticated watermelon survival strategies during the progression of drought (Nanasato et al. 2005). Protein abundance of cytochrome

stress, from a strategy for acquiring new water resources b561 increased in the leaf mesophyll cells of wild to that for preserving tissue water, by regulating its root watermelon under drought stress (Nanasato et al. 2005), proteome in a temporally programmed manner. suggesting that the activation of this redox chain may contribute to the excess energy dissipation under drought in the presence of strong light. Unique mechanisms for high light tolerance Photosynthetic apparatus in the thylakoid membranes in chloroplasts is one of the potential sites which are liable Accumulation of compatible solutes to be damaged under drought in the presence of high In response to water deficits, many plants synthesize and light. Under this excess light condition, some of the accumulate small organic compounds known as photosynthetic components in the chloroplasts tend compatible solutes in the cells. Compatible solutes are a to be over-reduced, which enhances the risks for group of compounds which do not interfere with cellular photoinhibition (Anderson and Barber 1996). Survival metabolisms at high concentrations (Hare et al. 1998; strategies of plants against excess light stress are diverse, Rontein et al. 2002). Compatible solutes are highly which include light avoidance by leaf movement or hydrophilic and include amino acids and their derivatives rolling (Larcher 1995), increase in the light reflection of such as proline, glycine betaine and citrulline, and the leaves by deposition of cuticle waxes (Cameron et al. carbohydrates such as sucrose, trehalose, mannitol, 2006), shielding from the excess photons by anthocyanin pinitol, fructan and raffinose. It has been proposed that accumulation (Gould 2004), thermal dissipation of these compounds play a role in osmotic adjustment of excess light energy by non-photochemical quenching in the cells under water deficits. However, transgenic plants photosystem II (Müller et al. 2001), and fortification of which accumulate them only at lower concentration often ROS scavenging system (Smirnoff 1993). displayed significant resistance to stress (Tarczynski et

Retama raetam (Figure 1F) is a stem-assimilating C3 al. 1993; Kishor et al. 1995), suggesting that these xerophyte that thrives in the Mediterranean region. This compatible solutes may have other functions in addition species contains two different populations of stems in a to the osmotic adjustment. A variety of protective roles single plant: although those of the lower canopy are have been suggested for the compatible solutes, such as shaded from direct sunlight and maintain capability for stabilization of cellular proteins and membranes, storage photosynthesis, the stems of the upper canopy are of nitrogen and/or reducing equivalent, and photosynthetically inert and establish tissue dormancy detoxification of ROS (Smirnoff and Cumbes 1989; Hare under drought conditions. Many of the essential proteins et al. 1998; Rontein et al. 2002). related to photosynthesis, such as RuBisCO, ascorbate Hydroxyl radicals are one of the most toxic agents peroxidase and D1 subunit of photosystem II are among known ROS, which react with various molecules virtually absent in the upper stems (Mittler et al. 2001). in the cells at diffusion-limited rates, and leads to the However, mRNAs for these proteins are stored in dysfunction of the cellular activities. The in vitro studies association with polysomes in the cells, and are ready for have shown that efficiency of compatible solutes for the rapid synthesis of these proteins when resuming detoxifying hydroxyl radicals is higher in polyol such as photosynthesis after rainfall. These observations suggest mannitol compared to proline and glycine betaine a presence of highly developed adaptive mechanisms in (Smirnoff and Cumbes 1989). The in vivo role of this plant, which protect the entire plant in the times of compatible solutes as ROS scavenger has been analyzed drought/high light stresses, and enable this plant to using transgenic plants in which polyol metabolism was compete with other plant species for the limited water modified. Transgenic tobacco in which E. coli mannitol- resources in the times of short rainy periods. 1-phosphate dehydrogenase was targeted to chloroplasts Another unique mechanism suggested in xerophytes by the addition of chloroplast transit peptide has been

involves a redox chain mediated by cytochrome b561 in generated (Shen et al. 1997). Mannitol accumulated the plasma membrane and ascorbate oxidase in the at an estimated concentration of up to 100 mM in the

apoplasts (Nanasato et al. 2005). Cytochrome b561 chloroplast of the transgenic plant, which showed catalyzes oxidation of ascorbate in the cytosol while increased tolerance to photooxidative treatment such as regenerating it in the apoplasts, thereby mediating trans- an application of methyl viologen (Shen et al. 1997). membrane electron transfer from the cytosol to the Therefore, it has been suggested that accumulation of apoplasts (Asard et al. 2001). Ascorbate oxidase, which polyol is one effective strategy to protect plant cells from catalyzes oxidation of ascorbate in the apoplast to yield photooxidative damages under drought in the presence of K. Akashi et al. 261

Figure 2. Pathways of citrulline metabolisms. Enzymes that catalyze the indicated reactions are: 1, N-acetylglutamate synthase; 2, N- acetylglutamate kinase; 3, N-acetylglutamate 5-phosphate reductase; 4, N-acetylornithine aminotransferase; 5, glutamate N-acetyltrasnferase; 6, carbamoylphosphate synthetase; 7, ornithine carbamoyltransferase; 8, argininosuccinate synthetase; 9, argininosuccinate lyase; 10, arginase; 11, urease. Molecular structure of citrulline is shown in inset. strong light. (Garwe et al. 2003). Integrative analyses on the In the leaves of wild watermelon, citrulline physiological, biochemical and molecular responses of accumulates up to 25 mmol g1 FW during drought in the wild plants under adverse environments will be an presence of high light (Kawasaki et al. 2000). Second- important step in unraveling the potentials of plant order rate constant of the reaction between citrulline and genetic resources on earth. Information from these hydroxyl radicals was shown to be 3.9109 M1 s1, studies is expected to reveal a wealth of genes that would demonstrating that citrulline is one of the most efficient be useful in molecular breeding of crop plants in the scavengers for hydroxyl radicals among known future. compatible solutes (Akashi et al. 2001). These observations suggested that hydroxyl radicals generated in the cells are efficiently decomposed in vivo, with a References calculated half time of approximately 0.9 ns. Citrulline is Adams P, Nelson D, Yamada S, Chmara W, Jensen RG, Bohnert an intermediate compound in the arginine biosynthetic HJ, Griffiths H (1998) Growth and development of pathway, in which 11 different enzymes are involved in Mesenbryanthemum crystallinum (Aizoaceae). New Phytol its metabolism (Slocum RD 2005; Figure 2). Recent 138: 171–190 study has indicated the intricate regulatory network in Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors the citrulline metabolism of this plant under drought and activates wax biosynthesis, alters cuticle properties, and confers high light stress (Takahara et al. 2005). drought tolerance when overexpressed in Arabidopsis. Plant Cell 16: 2463–2480 Akashi K, Miyake C, Yokota A (2001) Citrulline, a novel Wild plant genetic resources—Perspectives compatible solute in drought-tolerant wild watermelon leaves, is Terrestrial plants are overwhelmingly diversified not only an efficient hydroxyl radical scavenger. FEBS Lett 508: 438– for their morphology and life forms, but also for their 442 responses to any kind of external stimuli. For example, Akashi K, Nishimura N, Ishida Y, Yokota A (2004) Potent hydroxyl radical-scavenging activity of drought-induced type-2 there is a significant difference in the transcriptional metallothionein in wild watermelon. Biochem Biophys Res regulation during drought stress between wild plants and Com 323: 72–78 domesticated/model plants. M. crystallinum showed Anderson B, Barber J (1996) Mechanisms of photodamage and drought-induction of a gene for an enzyme, myo-inositol protein degradation during photoinhibition of photosystem II. In: 1-phosphate synthase, which involves in the committing Baker NR (ed). Photosynthesis and the environment. Kluwer step of the biosynthetic pathway for a compatible solute Academic Publishers, Dordrecht, The Netherlands, pp 101–121 pinitol (Ishitani et al. 1996). However, homologous gene Asard H, Kapila J, Verelst W, Berczi A (2001) Higher-plant plasma membrane cytochrome b : a protein in search of a function. in Arabidopsis was not induced during drought. A 561 Protoplasma 217: 77–93 similar phenomenon has been observed in many other Bartels D, Salamini F (2001) Desiccation tolerance in the wild plants such as wild watermelon (Akashi et al. resurrection plant Craterostigma plantagineum. A contribution 2004), Lycopersicon pennellii (Mittova et al. 2002), to the study of drought tolerance at the molecular level. Plant Tortula ruralis (Oliver et al. 2004) and Xerophyta viscosa Physiol 127: 1346–1353 262 Wild plant resources for drought tolerance

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