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First published online September 16, 2005; 10.1104/pp.105.065458 Plant Physiology 139:836-846 (2005) © 2005 American Society of Plant Biologists A Lily ASR Protein Involves Abscisic Acid Signaling and Confers Drought and Salt Resistance in Arabidopsis1,[w]Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227 (C.-Y.Y., Y.-C.C., C.-S.W.); and Institute of Botany, Academia Sinica, Taipei, Taiwan 11529 (G.Y.J.)
LLA23, an abscisic acid-, stress-, and ripening-induced protein, was previously isolated from lily (Lilium longiflorum) pollen. The expression of LLA23 is induced under the application of abscisic acid (ABA), NaCl, or dehydration. To provide evidence on the biological role of LLA23 proteins against drought, we used an overexpression approach in Arabidopsis (Arabidopsis thaliana). Constitutive overexpression of LLA23 under the cauliflower mosaic virus 35S promoter confers reduced sensitivity to ABA in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Transgenic 35S::LLA23 seeds are able to germinate under unfavorable conditions, such as inhibitory concentrations of mannitol and NaCl. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Thus, our results provide strong in vivo evidence that LLA23 mediates stress-responsive ABA signaling. In vegetative tissues, it is intriguing that Arabidopsis 35S::LLA23 stomata remain opened upon drought, while transgenic plants have a decreased rate of water loss and exhibit enhanced drought and salt resistance. A dual function of the lily abscisic acid-, stress-, and ripening-induced protein molecule is discussed.
Abscisic acid (ABA) plays important regulatory roles in various aspects of plant growth and development throughout the plant life cycle, particularly in the ability to sense and respond to various unfavorable environmental stresses, including drought, salt, and cold stresses during vegetative growth (Leung and Giraudat, 1998  ; Finkelstein et al., 2002). In vegetative tissues, ABA mediates adaptive responses to abiotic environmental stresses. In particular, during drought, ABA promotes stomatal closure and prevents stomatal opening, thus reducing transpirational water loss. During late embryogenesis, ABA promotes the acquisition of desiccation tolerance and seed dormancy and inhibits seed germination (Marcotte et al., 1992; Rock and Quatrano, 1995; Koornneef et al., 2002). Substantial progress has been made in the characterization of the ABA signal transduction cascades (Bonetta and McCourt, 1998; Leung and Giraudat, 1998; Kang et al., 2002; González-García et al., 2003). Genes that respond to exogenous ABA applications have been identified (Skriver and Mundy, 1990), and secondary messengers, such as Ca2+, cyclic ADP-ribose, and inositol triphosphates, have been implicated in ABA-mediated responses (Wu et al., 1997; Pandey et al., 2000; Viswanathan and Zhu, 2002). ABA signaling appears to involve a complex network of both positively and negatively regulating components, including kinases, phosphatases, and transcriptional regulators (Finkelstein et al., 2002; Abe et al., 2003; González-García et al., 2003). However, many of the cellular components and genes involved in ABA reception and downstream transduction have not been well characterized. Recently, Cakir et al. (2003) reported a grape (Vitis vinifera) ABA-, stress-, and ripening-induced (ASR) protein acting as a downstream component of a common transduction pathway for sugar and ABA signals. Although hypotheses have been proposed concerning ASR function, the precise in planta function of the protein remains unclear (Carrari et al., 2004).
The ASR proteins are characterized as small and heat-stable proteins because of their strong hydrophilicity. ASRs have been reported to be members of the widespread class of hydrophilins, including the seed-specific late embryogenesis abundant (LEA) proteins (Dure et al., 1989
The expression of ASR genes varied in pattern and specificity in different species, such as the fruit of tomato, pummelo (Citrus maxima), apricot (Prunus armeniaca), and grape (Iusem et al., 1993 Here, we use an overexpression approach in Arabidopsis to provide evidence on the biological role of LLA23 proteins against dehydration. Constitutive expression of the lily ASR under the cauliflower mosaic virus 35S promoter displays a reduced sensitivity toward ABA during seed germination, dormancy, and stomatal closure. The LLA23-overexpressing plants display altered expression of ABA/stress-regulated genes. Additionally, 35S::LLA23 transgenic plants exhibit markedly enhanced drought and salt resistance. These results may suggest a dual role of LLA23, acting as a regulator as well as a protective molecule upon water deficit.
Growth Phenotypes of LLA23 Overexpression Plants To examine the protective function of LLA23 proteins, we used an overexpression approach. The coding region of LLA23 was fused to the cauliflower mosaic virus 35S promoter, and the construct (Fig. 1A) was used to transform Arabidopsis (ecotype Columbia [Col]) plants. T1 and T2 kanamycin-resistant lines were recovered. There were five T3 homozygous lines, of which two with higher LLA23 expression levels were selected for more detailed analysis. When grown in the absence of ABA, the 35S::LLA23 transgenic lines did not display any visible phenotypic alteration compared with the Arabidopsis wild-type plants of 2 weeks old (Fig. 1B). Northern analysis confirmed that the transcripts were present in leaf tissue of both transgenic plants harvested at 2 weeks, whereas no expression was detected in wild-type plants, as expected (Fig. 1C). The LLA23 protein was also determined in extracts from the same tissues. As shown in Figure 1D, the protein was detected in seedlings of both transgenic plants. No protein was detected in wild-type plants.
Constitutive Expression of LLA23 in Arabidopsis Reduced ABA Sensitivity in Seeds
The specific induction of LLA23 expression by ABA in lily pollen (Wang et al., 1998
When freshly released from the mother plant, Arabidopsis seeds display primary dormancy. Seeds are unable to germinate without the help of dormancy-breaking agents such as acclimation. To determine the degree of dormancy of 35S::LLA23 seeds, we compared the germination percentage of the seeds harvested at the same time after different acclimation periods (0, 48, and 96 h) with those of the wild-type plants and ABA-related mutants that produce nondormant (gin1-3) or weak dormant (abi4-1) seeds. As shown in Figure 3, the two 35S::LLA23 seeds exhibited a significantly reduced dormancy compared with the wild type. In the absence of acclimation at 4°C, 35S::LLA23 seeds were able to germinate, reaching 50% germination, which was very similar to that of abi4-1 mutants. It indicated that the two 35S::LLA23 transgenics exhibited weak dormancy; they have lost some, but not all, dormancy as a result of the LLA23 protein involved in modulating ABA signaling.
35S::LLA23 Seeds Exhibit Resistance to Salt and Osmotic Stresses
High concentrations of salt inhibit the germination of Arabidopsis (Werner and Finkelstein, 1995
While germination and growth of 35S::LLA23 seeds were affected significantly by 60 mM NaCl, their response to the same concentration or twice the concentration of mannitol (120 mM), which gave the same osmotic pressure, was normal (Fig. 4B). Thus, in addition to the effect of osmosis, reduced sensitivity of 35S::LLA23 seeds appeared to be ionic in nature.
The growth and development of LLA23-overexpressing plants in soil in the growth chamber without stress appeared normal, as shown in Figure 5, A and B. When the transgenic wild-type plants and an aba mutant, gin1-3, were sprayed with 5 µM ABA, it showed that >60% of 35S::LLA23 stomata became closed compared to 89% of both wild-type (116/131) and gin1-3 (102/115) stomata. Therefore, 35S::LLA23 plants can be properly induced by ABA, but their responses to ABA are not as effective as wild-type plants, indicating the involvement of LLA23 proteins in ABA signaling.
When the soil was allowed to dry by withholding water, 48% of both 35S::LLA23 plants and 84% of the wild-type plants became wilted 12 d after the withdrawal of water (Fig. 5C). The two 35S::LLA23 lines that remained upstanding did not begin to wilt until 16 d after the withdrawal of water (data not shown). When the wilted plants were rewatered afterward, only 5% of the wilted wild-type plants recovered, whereas >50% of both wilted 35S::LLA23 lines survived to maturity (Fig. 5D). To minimize variations, transgenic and wild-type plants were grown on soil in the same container and that also led to the same result (data not shown), in which 35S::LLA23 transgenic plants survived the drought conditions better than the wild-type plants. Corroborating data were obtained when the rate of water loss from detached leaves of wild-type and LLA23-overexpressing plants was compared. The leaves tested were of similar size and age. Leaves of transgenic plants overexpressing the LLA23 protein had lower rates of water loss than that of wild-type plants (Fig. 5E). Microscopic examination of stomatal opening of 35S::LLA23 leaves showed that approximately 86% (78/91) of 35S::LLA23E stomata remained open under drought stresses for 12 d, a percentage close to 96% (74/77) of the wild-type stomata without stress (Fig. 5F). The unexpected observation of stomatal opening was further checked by measuring the levels of ABA of both plant types. Correlated with the opening of 35S::LLA23 stomata, the transgenic plants did not appreciably increase their ABA levels upon drought stresses for 12 d, similar to that of the wild-type plants at normal growth conditions (Table I). In contrast, the level of ABA in wild-type leaves significantly increased approximately 10-fold under drought stresses as compared with the unstressed conditions. It clearly indicated that due to the presence of LLA23 proteins, the response to ABA was delayed in the transgenic plants upon water deficit. Instead, the LLA23 protein functions as a water-retaining molecule that confers drought resistance on 35S::LLA23 plants based on the drought test of transgenic lines in which leaves of these plants overexpressing LLA23 protein had lower rates of water loss than wild-type plants (Fig. 5E), while 35S::LLA23 stomata remained open upon drought stresses for 12 d. The protective concept of LLA23 as a water-retaining molecule is further in agreement with the osmotic potential assays in which 35S::LLA23 leaves show –1.30 and –0.97 MPa, respectively, the levels not appreciably changed upon drought stress for 12 d when compared with that in unstressed wild-type plants (Table I). Nevertheless, if these plants were continued to withhold water for additional 4 d, most 35S::LLA23E stomata (87%) became closed (Fig. 5F). Moreover, transgenic plants began to wilt and their ABA levels in leaves markedly increased, although the increase of ABA levels in 35S::LLA23 plants was reduced (Table I).
In addition to the marked differences of ABA level and stomatal change in opening/closure, transgenic plants at 12 and 16 d from water holding are apparently different in appearance. Most LLA23-overexpressing plants remain upstanding at 12 d, while all of them are wilted at 16 d from water holding. Moreover, leaves of LLA23-overexpressing plants remain green and normal at 12 d, while they turn yellowish and wrinkle 16 d after the withdrawal of water. Taken together, these results suggested that under drought stresses for 12 d, an ABA response was delayed in LLA23-overexpressing plants. The LLA23 proteins possess water-retaining ability that makes 35S::LLA23 plants not perceive drought stresses; consequently, the level of ABA in transgenic lines is kept as low as wild-type plants. Since the level of ABA did not increase in plants, their stomata remained open. It did not increase until severe drought conditions (16 d from water holding) were applied. However, the induced level of ABA in 35S::LLA23 plants at 16 d from water holding was lower than that in wild-type plants, also suggesting a potential influence of LLA23 proteins in ABA signaling.
Plants of 35S::LLA23 and wild type (Fig. 6A) also were examined in the growth chamber for resistance to NaCl. The growth and development were reduced in both wild-type and transgenic plants, but the reduction is more severe in wild-type plants than in transgenic lines when socked in a solution containing 400 mM NaCl every 3 d for 2 (Fig. 6B) and 3 weeks (Fig. 6C). Compared with wilted wild-type plants, 35S::LLA23E plants showed a reduced inhibition of plant growth and remained upstanding on the soil. 35S::LLA23C plants also displayed a similar phenotype to 35S::LLA23E plants after salt treatments (data not shown). The stem weight and length of both transgenic lines are heavier and longer than those of wild-type plants. Also, the number of siliques in both 35S::LLA23 transgenics is more than that of wild-type plants (Fig. 6D). Thus, these results demonstrate that both 35S::LLA23C and 35S::LLA23E transgenic plants exhibit significant resistance to salt.
Expression of ABA/Stress-Responsive Genes in 35S::LLA23 Plants
To examine the regulatory roles of LLA23 in planta, the expression of various ABA/stress-responsive genes in 35S::LLA23 plants was determined using quantitative RT-PCR (Q-PCR). With or without an addition of 5 µM ABA, the transcript levels of a number of ABA-regulated genes were enhanced in 35S::LLA23C and 35S::LLA23E transgenic lines compared to the wild-type plants. These include RD29b (Yamaguchi-Shinozaki and Shinozaki, 1994
In our previous reports (Wang et al., 1998 ; Huang et al., 2000), we described a lily-pollen-specific LLA23 cDNA that encodes a hydrophilic protein with a calculated molecular mass of 16 kD and a pI of 6.1. The lily LLA23 protein is a member of the ASR protein family. Although ASR proteins are present in various organs of a large variety of plant species (Cakir et al., 2003), no ASR homolog exists in Arabidopsis. The relatively different Mrs, specific localization within cells, and the presence of ASR in various organs may indicate diversities in function.
Signal transduction pathways that operate in a cell require positive and negative regulators for a proper control. In this study, there are several lines of evidence that strongly indicate LLA23 as a regulator. First, 35S::LLA23 seeds show reduced sensitivity to ABA. While the germination of wild-type Arabidopsis seeds is suppressed by 5 µM ABA, LLA23-overexpressing seeds emerge radicles under these conditions (Fig. 2). Second, the two 35S::LLA23 lines display a reduced dormancy compared with the wild type, very similar to that of the abi4-1 mutant (Fig. 3). Third, 35S::LLA23 seeds and plants exhibit resistance to salt, osmotic, and drought stresses (Figs. 4–6
The notion that LLA23 as a regulator is reinforced by the demonstration of nuclear-targeting of LLA23 by its NLS (Wang et al., 2005 ). A grape ASR protein was reported to act as part of a transcription-regulating complex involved in ABA and sugar signaling (Cakir et al., 2003). It is correlated with a recent report where tomato ASR1 is likely involved in the regulation of several water- or salt-stress-modulated gene expression (Kalifa et al., 2004b). Currently, we have found that the transgenic plants overexpressing LLA23 reveal reduced sensitivity to 6% Glc (C.Y. Yang, Y.C. Chen, and C.S. Wang, unpublished data). Thus, the LLA23-overexpressing plants related to sugar signaling should warrant further investigation.
Arabidopsis transgenic lines overexpressing LLA23 confer improved resistance to water deficit and salt (Figs. 5 and 6). It is rationale to suggest that 35S::LLA23 plants possess two levels of protection against drought. The ground level of protection comes from the high hydrophilicity of the LLA23 protein that may display enhanced water-retaining ability. The water-retaining ability of LLA23 is also reflected by the fact that 35S::LLA23 leaves have a little change of osmotic potential 12 d after the withdrawal of water (Table I). Other osmoprotective molecules possibly induced upon drought may also exist in the transgenic plants. In fact, a number of ABA/stress-responsive genes, such as RD29b and KIN2, are up-regulated in 35S::LLA23 plants (Fig. 7). Additionally, the presence of LLA23 proteins in abundance in the maturing pollen of lily plants as well as in 35S::LLA23 plants (Fig. 1D) corroborates the concept that LLA23 acts as a protective molecule. Like LLA23, LEA proteins have been hypothesized to play a potential protective role under unfavorable environments based on their high average hydrophilicity. Constitutive overexpression of LEA proteins reported by several studies exhibits a significant increase in their tolerance to dehydration, salt, or freezing conditions (Xu et al., 1996
The secondary level of protection comes from the regulatory properties of LLA23 proteins as described previously. The expression of RD29b and KIN2 is up-regulated in 35S::LLA23 plants, suggesting that LLA23 mediates stress-responsive ABA signaling. The LLA23 protein that acts as a regulatory factor is also supported by a recent report in which the ASR protein is recognized as a transcription factor (Carrari et al., 2004
When the soil is dried for 12 d, a rising level of ABA does not occur (Table I). Consequently, 35S::LLA23 stomata remain open at 12 d after the withdrawal of water. As 35S::LLA23 plants appear drought resistant, the response to ABA at the meantime is delayed in these plants. Thus, it results in a seemingly contradictory observation that 35S::LLA23 stomata remain open upon drought. Since ABA does not increase at 12 d from water holding, the transcript levels of those genes examined do not appreciably change (Fig. 7), suggesting that the drought-resistant property of 35S::LLA23 plants is likely attributed to the water-retaining ability of LLA23.
If dehydration goes worse, would the LLA23-overexpressing plants perceive drought stresses? As expected, when the soil is allowed to withhold water for an additional 4 d, they begin to respond to water deficit and their ABA levels are markedly induced in transgenic leaves, resulting in stomatal closure (Fig. 5F). The increase of ABA levels also changes the expression patterns of several genes examined and that alteration is mediated by LLA23 in 35S::LLA23 plants (Fig. 7). However, the level of ABA in both 35S::LLA23 leaves is somewhat reduced at 16 d from water holding (Table I). The reduced ABA inducibility may be attributed to the regulation of LLA23 in ABA signaling. The reduction of ABA sensitivity occurs not only in plants but in seeds of 35S::LLA23 transgenics. Constitutive expression of LLA23 in transgenic seeds significantly reduced ABA sensitivity during dormancy and germination tests of various unfavorable conditions (Figs. 2–4
Plant Growth and Inoculation Arabidopsis (Arabidopsis thaliana) plants (Col ecotype) were used in this study. gin1-3, an ABA-deficient mutant, was obtained from Dr. W.-H. Cheng (Institute of Botany, Academia Sinica, Taipei, Taiwan) and abi4-1, an ABA-insensitive mutant, from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH), and their phenotypes were confirmed before use. Wild-type and mutant seeds (at least 50 seeds each) were sterilized and kept for 4 d at 4°C in the dark to break dormancy. Seeds were grown at 22°C under long-day conditions (16-h-light/8-h-dark cycle) aseptically or on soil. For soil growth, seed were sown on a 1:1:8 mixture of vermiculite, perlite, and peat moss irrigated with water and transferred to normal growth conditions. Unless stated otherwise, the plants were watered every other day.
To generate the construction, the LLA23 cDNA was amplified by PCR using pLLA23 cDNA (Huang et al., 2000
Wild-type and mutant seeds (at least 50 seeds each) were aseptically treated with 70% ethanol for 5 min and then with 10% household bleach for 30 min, washed four times with sterile water, and water imbibed in the dark for 3 d at 4°C. To measure ABA sensitivity, seeds were then sown directly on the surface of filter papers soaked with MS solution containing 1x Murashige and Skoog basal salt mixture (Murashige and Skoog, 1962
For drought treatment, 3-week-old soil-grown plants were withheld completely from water for 12 d. The number of wilted plants was scored and is expressed as the percentage of the total plants. Wilted plants then were given water again (on day 13), and the number of recovered plants that fully regained turgor and resumed growth was scored after an additional 4 d (day 16) and is expressed as the percentage of the total plants wilted. For salt treatment, 1-month-old plants grown in each pot (250 cm3) were socked in 400 mM NaCl solution (200 mL per pot) freshly prepared every 3 d, and plants were continuously socked for 2 or 3 weeks. Shoot fresh weight and length and silique number after salt treatment were counted. Ten plants of each genotype and three replicates each were used. Photographs were taken at the end of treatment.
To examine guard cells, leaves excised from wild-type and transgenic lines with or without drought treatment were placed on slides abaxial side up immediately after excision, and photographs were taken. Rosette leaves were excised from wild-type and transgenic lines under drought treatment as described above. ABA measurement is according to the method of Xiong et al. (2001)
Total RNA was extracted from leaves of Arabidopsis using the Ultraspec RNA isolation system (Biotecx Laboratories). RNA samples were electrophoresed in 1.0% formaldehyde-MOPS gels using standard procedures (Sambrook et al., 1989
For real-time Q-PCR, the cDNA was amplified in the presence of SYBR Green I Nucleic Acid Stain (Cambrex 50513) 10,000x dilution from stock using a Rotor-Gene 3000 (Corbett). Amplification of actin cDNA under identical conditions was used as an internal control to normalize the level of cDNA. The data obtained were analyzed with Rotor-Gene 6 software (Corbett). Since SYBR Green I dye binds to the minor groove of any double-stranded DNA, including specific products, nonspecific products, and primer dimers, it is necessary to perform a melting curve analysis at the end of each Q-PCR experiment. Nonspecific products or primer dimers can be identified as they melt at a lower temperature compared to the specific amplicon. Specific temperatures obtained for ACTIN (89°C), KIN2 (91°C), RD29b (88°C), COR15a (88°C), P5CS1 (88°C), and ADH1 (88°C) validated the specific product formation. Primers used in the Q-PCR reactions are listed in Table II. Q-PCR experiments were repeated three times independently, and the data were averaged.
Protein Preparation, Electrophoresis, and Immunoblotting
Phenol extraction of total protein was performed according to Wang et al. (1992) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AF077629.
We greatly thank Dr. W.-H. Cheng (Institute of Botany, Academia Sinica, Taipei, Taiwan) for critical reading of the manuscript. Received May 11, 2005; returned for revision August 8, 2005; accepted August 11, 2005.
1 This work was supported by the National Science Council of the Republic of China under grant NSC93–2311–B–005–007 to C.-S.W. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.065458. * Corresponding author; e-mail cswang2{at}dragon.nchu.edu.tw; fax 886–4–2285–3527.
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ABSTRACT
RESULTS
(1)-pyrroline-5-carboxylate synthetase 1 (P5CS1) did not appreciably change in 35S::LLA23 plants, suggesting that the synthesis of Pro in transgenic lines remained the same as wild-type plants. P5CS1 is the rate-limiting enzyme in the biosynthesis of Pro (Yoshiba et al., 1999
