Index References

Sessile plants have been evolved to adapt to variety of environmental stimuli by different strategies from those of animals. In plants, regulation of gene expression at the level of transcription appears to be a key means of control in the responses to the environment. It has been proposed that alterations in the expression of genes coding for transcriptional regulators are a major source of diversity that underlie evolution (Carroll, 2000). In Arabidopsis thaliana, about 45% of transcription factors are those of types that are found only in plants (Riechmann et al., 2000). Therefore, it is highly likely that some of these plant-specific transcription factors have evolved for the regulations of responses to environmental changes. RAV1 and RAV2 identified in A. thaliana are novel type of transcription factors, containing two distinct DNA-binding domains, AP2/EREBP and B3, both of which are found only in higher plants (Kagaya et al., 1999a; Giraudat et al., 1992; Suzuki et al., 1997; Riechmann and Meyerowitz, 1998; Riechmann et al., 2000). Previous studies have shown that the expression of RAV1 and RAV2 was stimulated by various external or environmental cues, which include low temperature, darkness, wounding, drought and salt stress, and pathogen attack (Fowler and Thomashow, 2002; Lee et al., 2005; Sohn et al., 2006). Thus, the two transcription factors may be implicated in the adaptation to variety of environmental stimuli. In A. thaliana, there are a subset of genes, known as touch-induced (TCH) genes, that are rapidly upregulated following a variety of external stimuli such as touch, wind, cold shock, darkness, wounding and fungal elicitors (Antosiewicz et al., 1995; Braam, 1992; Braam and Davis, 1990). The diverse stimuli capable of inducing TCH gene expression appear to share a common property of causing mechanical perturbations, perhaps at a cellular level (Braam, 2000). A variety of environmental stimuli are known to cause alteration in plant growth pattern. For example, A. thaliana plants stimulated by touch develop shorter petioles and bolt (Braam and Davis, 1990). Such developmental responses are known as thigmomorphogenesis (Jaffe, 1973). Plant hormones and other signaling molecules such as octadecanoids, ethylene, intracellular calcium ions and reactive oxygen species have been implicated in touch responses (reviewed by Braam, 2005). Furthermore, a genome-wide search revealed that touch-induced gene expression is widespread; more than 2.5% of A. thaliana genes are rapidly upregulated in response to touch-stimulus (Lee et al., 2005). Among these genes, overrepresented are those encoding calcium-binding proteins, enzymes for cell wall modification, proteins involved in defense, transcription factors and protein kinases (Lee et al., 2005). Despite the catalogs of the signaling molecules and the touch-induced genes, however, the mechanisms that underlie thigmomorphogenesis are largely unknown.

In our previous study, the expression of RAV1 and RAV2 were found to be modulated by simply transferring plants to distilled water (Kagaya et al., 1999b). However, the true nature of the stimulus accomapnied by such a treatment was not clear. In this report, we analyzed the expression of RAV1 and RAV2 in response to various external stimuli and found that the expression of RAV1 and RAV2 was regulated by touch-related mechanical stimuli with temporal expression patterns distinct from those of the known TCH genes. Based on these observation, possible roles of RAV1 and RAV2 in developmental regulation in response to environmental stimuli are discussed.

Seedlings of A. thaliana (ecotype, Columbia) were grown on agar medium plates containing half-strength MS salts (Murashige and Skoog, 1962) and 1% sucrose at 22°C under continuous light. Two to three-week-old plants were used for experiments. The rids of the plates were always kept on except for very short time periods when giving mechanical stimuli. Extraction of RNA from frozen materials and northern blot hybridization were carried out as described previously (Yamaguchi-Shinozaki and Shinozaki, 1994). Gene-specific hybridization probes for RAV1 and RAV2 were prepared from the previously described full-length cDNA clones (Kagaya et al., 1999a). Probes for TCH3 (Sistrunk et al., 1994) and TCH4 (Xu et al., 1995) were obtained by amplifying the coding region of these genes from A. thaliana genomic DNA. The primers used were as follows: TCH3, 5’-GTTACATTACCGTGAATGAGCTCCGTA-3’ and 5’-TCAAGATAACAGCGCTTCGAACAAAT-3’; TCH4, 5’-TTGAAATCACCTGGAACAACATGGGA-3’ and 5’-CTATGCAGCTAAGCACTCTTTAGGAAGA-3’. A probe for GUS was prepared from pBI221 (Clontech, Palo Alto, CA, USA). The probe DNA fragments were radiolabeled with ³²P using a commercial random labeling system (Amersham, Buckinghamshire, UK). Transgenic A. thaliana plants carrying a chimeric gene (RAV2-GUS) consisting of a 1.8 kbp RAV2 promoter fragment and the GUS coding region were described previously (Kagaya et al., 1999b). The T₃ plants derived from five independent transformed lines were used for northern blot and GUS activity analyses. Histochemical staining for GUS activity was carried out as described by Jefferson et al. (1987). After the staining, plants were immersed twice in 70% ethanol at 37°C overnight to fix and to remove chlorophyll.

Several different treatments were tested to clarify the nature of stimuli which caused an induction of RAV1 and RAV2 expression. Time course samples (0, 1, 3 or 6 hr) were collected from plants on each plate after the treatment, and used to measure relative levels of RAV1 and RAV2 transcripts by northern blot analysis (Fig. 1(A)). As previously reported, by simply transferring plants to distilled water, the levels of RAV1 and RAV2 transcripts were transiently increased at 1 hr and then decreased at 3 hr (Fig. 1(A), "Transfer"). A pulse of water spraying (5 squirts) resulted in a similar transient increase in the levels of RAV1 and RAV2 transcripts with a peak level at 1 hr (Fig. 1(A), "Spray"). Although a contact with water was common to the two treatments, it was not the nature of the stimulus that elicited the transient upregulation of RAV1 and RAV2 because the increases in the transcript abundance were also observed after an exposure to gusts of wind (Fig. 1(A), "Wind"). Gentle touches with a finger of the rosette leaves again resulted in an induction of expression of RAV1 but not RAV2 at 1 hr after treatment in this experiment (Fig. 1(A), "Touch"). However, the experiment with a shorter time range revealed transient increases in both RAV1 and RAV2 transcripts at 15 min after touch-stimulus (Fig. 1(B)). These results indicated that mechanical perturbation of plants was the fundamental nature of the stimuli that caused transient upregulation of the two genes. Such regulation resembled with that of the previously described TCH genes. However, from a closer examination of the results, we noticed that the expression levels of RAV1 and RAV2 increased again following the transient upregulation at 6 hr after the transfer treatment (Fig. 1(A), "Transfer"). In "touch" or "wind"-treated plants, higher levels of the RAV2 expression were observed at 6 hr compared to that at 0 hr (Fig. 1(A), "Touch" and "Wind"). Thus, we examined the induction kinetics of the RAV1 and RAV2 expression in detail (Fig. 2). Here, we adopted the "spray" treatment as inductive stimulus because it has been popularly used to study TCH genes, was easiest to control and gave most reproducible results among the tested treatments. Upregulation of RAV1 and RAV2 in response to "spray" was already marked at 15 min, reached to the maxima at 30 min. At 2 hr after the treatment, the expression levels of the two genes returned to the basal levels. However, at 3 hr, increases in the levels of RAV1 and RAV2 transcripts were again observed, and the increased levels were sustained up to 12 hr. Such a biphasic upregulation of RAV1 and RAV2 in response to mechanical stimuli contrasted to the responses of typical TCH genes such as TCH3 and TCH4 (Sistrunk et al., 1994; Xu et al., 1995). The TCH3 transcript level was also already higher at 15 min than at 0 min, continued to be increased up to 1 hr and gradually decreased to the basal level thereafter (Fig. 2). On the other hand, an increased expression of TCH4 in response to the stimuli was highly transient, which was only observed at 15 min. However, the induction of neither of them was biphasic as in the case with RAV1 and RAV2.

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Fig. 1 Time course analysis of RAV1 and RAV2 gene expression induced in response to different stimuli. Total RNAs isolated from plants harvested at the indicated times [(A) 0, 1, 3 and 6 hr; (B) 0, 15 and 30 min] after the indicated treatments were analyzed by northern blot hybridization using 32P-labeled probes specific to the indicated genes. Ethidium bromide (Et-Br)-stained rRNA bands were used to verify RNA loading and quality. Treatments were conducted as follows: “Transfer”, plants were removed from the media and placed in a 10 cm petri dish containing 20 ml distilled water; “Spray”, plants were sprayed with 5 squirts of distilled water onto the plants; “Wind”, plants were blown by a hair dryer for 1 min with a cool setting; “Touch”, rosette leaves were gently touched by placing a bare finger.

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Fig. 2 Induction kinetics of RAV1 and RAV2 in response to "spray"-stimulus are distinct from those of TCH3 and TCH4. Total RNAs isolated from plants harvested after "spray" treatment as described in Fig. 1 were analyzed by northern blot hybridization. Details of the experiment and presentation are as in Fig. 1. The minor transcripts just below the major band of RAV2 are suspected to represent those with different polyadenylation sites because cDNA clones for such transcripts are recorded on the databases (e.g. TAIR; http://www.arabidopsis.org/).

To test whether the expression levels of the two genes in the second phase of the biphasic induction were affected by the dose of the stimulus, the transcript levels were compared between plants received the standard and 5-times-intense spray stimulus (Fig. 3). The increased dose of the stimulus in fact resulted in higher levels of RAV1 and RAV2 expression not only at 1 hr but also at 6 hr after the stimulus. Thus, the levels of both the primary and the second phase upregulation of RAV1 and RAV2 by mechanical stimulus were likely to depend on the dose of the stimulus. At 3 hr, the plants received a higher dosage of the stimulus showed obvious downregulation of RAV1 and RAV2 to the initial or further lower levels after the primary transient, which could only be detected at an earlier time point in the plants received the standard dosage as seen in Fig. 2. This indicated that the intensity of the initial stimulus affected not only the levels but also the temporal patterns of response.

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Fig. 3 Dose-dependence of RAV1 and RAV2 induction by mechanical stimulus. Total RNAs isolated from plants harvested at indicated times after "spray" treatment as in Fig. 1 (x1) or with 5 times more squirts (x5), were analyzed by northern blot hybridization. Details of the experiment and presentation are as in Fig. 1.

We previously reported the spatial patterns of RAV2 expression using transgenic A. thaliana plants carrying RAV2-GUS in which the GUS coding region was placed downstream of a 1.8 kb RAV2 promoter fragment. We here asked whether this upstream region of RAV2 was sufficient to confer the rapid and transient RAV2 induction in response to mechanical stimuli. We found that the GUS transcript levels increased within 15 min and reach to the maximum at 30 min after spray treatment (Fig. 4(A)). The kinetics of the GUS induction was very similar to that of RAV2 measured with the same RNA samples (Fig. 4(A)). The results clearly indicated that the mechanical stimulus-induced RAV2 upregulation was controlled at the level of transcription and that the 1.8 kb promoter was sufficient for the regulation. The GUS transcripts already declined at 1 hr and returned to a near-basal level at 2 hr. Thus, the transcriptional activation was also transient. However, the downregulation of RAV2 was more rapid; the transcripts were decreased to the basal level already at 1 hr in this experiment. The rapid decay of RAV2 transcripts relative to that of GUS indicated that the RAV2 transcript has a higher turnover rate, as is known for other touch-induced genes (Gutiérrez et al., 2002). It would be interesting to address in the future whether the regulation of RNA stability would play any roles in the second phase upregulation of RAV2 in response to mechanical stimuli.

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Fig. 4 Spatial and temporal expression patterns of the GUS reporter gene directed by the RAV2 promoter in transgenic plants. (A) Total RNAs isolated from the RAV2-GUS transgenic plants harvested after "spray" treatment as in Fig. 1 were analyzed by northern blot hybridization. Details of the experiment and presentation are as in Fig. 1. (B) Histochemical GUS staining of the RAV2-GUS transgenic plants after the indicated times of "spray" treatment as described in Fig. 1. "C" indicates cotyledon; Bar = 1 cm.

We also examined whether any specific parts of plants respond to mechanical stimuli with regard to RAV2 upregulation using the RAV2-GUS plants (Fig. 4(B)). Without mechnical-stimuli, GUS expression was detected in the roots and the vascular tissues of cotyledons and rosette leaves. In rosette leaves, irregular and spotty patterns of staining were also observed before treatment. Two hours after spray-stimuli, drastic changes in the distribution of GUS expression were observed. Prominent GUS staining was observed in the leaf primordia and shoot apical meristems. In rosette leaves, particularly in petioles, uniform staining was observed (Fig. 4(B)). The intensities of staining were higher in young leaves than in older leaves. In the cotyledons, no obvious changes of staining patterns and intensities were detected (Fig. 4(B)). The distribution of GUS expression at 9 h was similar to that at 2 h (data not shown).

Previous studies have shown that A. thaliana plants stimulated by touch develop shorter petioles and bolts, and delay in flowering (Braam and Davis, 1990). Because GUS activities accumulated in the petioles and shoot apical meristems at high level in response to mechanical stimuli, RAV2 may function in such negative regulation of the growth and development in response to touch-related stimuli. In accordance with such a hypothesis, the transgenic A. thaliana plants of overexpressing RAV1 have been reported to show growth retardation represented by less numbers of lateral roots and rosette leaves, and delayed flowering (Hu et al., 2004). Considering the high similarities in the sequences as well as the temporal expression patterns between RAV1 and RAV2, RAV2 is also likely to function as a negative regulator of growth and development. Touch-induced morphological changes are known as thigmomorphogenesis that occurs slowly over time (Jaffe, 1973). However, the key regulators that lead to thigmomorphogenesis are not known. In the present work, we showed that the genes for plant specific transcription factors, RAV1 and RAV2, were upregulated by touch-related mechanical stimuli in transient, biphasic and dose-dependent manners. To date, no other TCH genes have been known that show such biaphasic induction with sustained upregulation after the transient induction. It would be interesting to hypothesize that RAV1 and RAV2 may function in the control of long term responses to mechanical stimuli such as thigmomorphogenesis. The sustained RAV2 upregulation in petioles and shoot apical meristems is in agreement with such a hypothesis.

The authors wish to thank Drs. Akiko Yamamoto and Yasuo Kowyama for helpful suggestions.