- Split View
-
Views
-
Cite
Cite
Yusuke Saijo, Natsuko Kinoshita, Keiki Ishiyama, Shingo Hata, Junko Kyozuka, Toshihiko Hayakawa, Teiji Nakamura, Ko Shimamoto, Tomoyuki Yamaya, Katsura Izui, A Ca2+-Dependent Protein Kinase that Endows Rice Plants with Cold- and Salt-Stress Tolerance Functions in Vascular Bundles, Plant and Cell Physiology, Volume 42, Issue 11, 15 November 2001, Pages 1228–1233, https://doi.org/10.1093/pcp/pce158
- Share Icon Share
Abstract
A rice Ca2+-dependent protein kinase, OsCDPK7, is a positive regulator commonly involved in the tolerance to cold and salt/drought. We carried out in situ detection of the transcript and immunolocalization of the protein. In the wild-type rice plants under both stress conditions, OsCDPK7 was expressed predominantly in vascular tissues of crowns and roots, vascular bundles and central cylinder, respectively, where water stress occurs most severely. This enzyme was also expressed in the peripheral cylinder of crown vascular bundles and root sclerenchyma. Similar localization patterns with stronger signals were observed in stress-tolerant OsCDPK7 over-expressing transformants with the cauliflower mosaic virus 35S promoter. The transcript of a putative target gene of the OsCDPK7 signaling pathway, rab16A, was also detected essentially in the same tissues upon salt stress, suggesting that the OsCDPK7 pathway operates predominantly in these regions. We propose that the use of the 35S promoter fortuitously strengthened the localized expression of OsCDPK7, resulting in enhancement of the stress signaling in the inherently operating regions leading to improved stress tolerance.
(Received May 23, 2001; Accepted August 27, 2001).
Introduction
In plants, Ca2+ signaling has been shown to be involved in multiple cellular responses to a wide range of environmental stimuli, including abiotic stresses such as cold, high salinity, and drought (Sanders et al. 1999). It is presumed that Ca2+-dependent protein kinases (CDPKs) play important roles in the regulation of this Ca2+ signaling (Harmon et al. 2000, Sanders et al. 1999). These kinases, currently known only in plants and some protista, contain an intrinsic calmodulin-like regulatory domain with four Ca2+-binding EF hands on each C-terminal side. CDPKs are activated directly through Ca2+ binding to these sites. The large number of CDPK isoforms in a given plant species provides the basis for the specificity and flexibility of the Ca2+ signaling, possibly through different expression patterns and/or distinct enzymatic properties (Hong et al. 1996, Lee et al. 1998). Therefore, it is very important to clarify the specific mode of action of each CDPK isoform. Our previous study showed that over-expression of a rice CDPK, OsCDPK7, driven by the cauliflower mosaic virus (CaMV) 35S promoter confers both cold- and salt/drought-stress tolerance on rice plants (Saijo et al. 2000). With the exception of this case, little is known about the physiological function of a particular CDPK pathway at present.
The cell-type specificity of changes in the cytosolic free Ca2+ concentration under abiotic stresses (Kiegle et al. 2000) implies that Ca2+ signal transduction leading to stress tolerance in plants differs among functionally different cell types. To further elucidate the molecular mechanism underlying OsCDPK7-mediated stress tolerance, the localization of this CDPK protein is one of the key issues that should be addressed. Although other researchers have reported that manipulation of protein kinases and transcription factors successfully improved multiple stress tolerance of plants (Jaglo-Ottosen et al. 1998, Kovtun et al. 2000, Liu et al. 1998), the tissue localization of the stress signal transduction remains to be examined.
It was found that the expression of stress-responsive genes encoding highly hydrophilic polypeptides such as late-embryogenesis-abundant (LEA) proteins is very high in the vascular transition area, where a water-deficit appears to occur most severely upon cold and salt/drought stresses (Danyluk et al. 1998, Garcia et al. 1998, Houde et al. 1995, Ono et al. 1996, Pearce et al. 1998). Notably, in the OsCDPK7 over-expressing transformants, high-level induction of the genes for LEA proteins occurred in response to high salinity and drought (Saijo et al. 2000).
Here, we investigated localization of the mRNA and enzyme protein of OsCDPK7 in the crowns and roots of wild-type and OsCDPK7 over-expressing rice plants. We also localized the transcript of the rab16A gene for a LEA protein. The results suggest that the OsCDPK7 pathway operates predominantly in vascular tissues, and that enhancement of the signaling in the proper regions leads to both cold- and salt-stress tolerance in the transformants.
Results
Localization of the OsCDPK7 mRNA and protein in rice crowns
The cereal crown is a heterogeneous organ consisting of various parts, which differ markedly in abitotic stress sensitivity (Olien 1967, Pearce et al. 1998). In addition, the stress tolerance of this organ is crucial for survival of the plant as a whole. Therefore, we first concentrated our attention on crowns to localize OsCDPK7 mRNA expression in the above-ground portions of rice plants. Hybridization of crown sections, derived from wild-type rice seedlings that had been exposed to cold stress, with a digoxigenin-labeled antisense OsCDPK7 probe revealed intense signals for OsCDPK7 expression most highly in vascular bundles, and the peripheral cylinder of vascular bundles (Fig. 1A, B). Southern blot analyses of rice genomic DNA with this OsCDPK7 probe revealed a single band, confirming that this probe is specific for the gene (data not shown). In contrast, other tissues, e.g. the crown cortex and mesophyll cells in the initials of leaf sheaths, exhibited very low signals. Signals were below detectable levels in any tissues of rice seedlings under salt stress or the normal growth conditions (data not shown). Our previous work showed that the OsCDPK7 mRNA level is significantly high under salt stress, although it is the highest under cold stress (Saijo et al. 2000). Further experiments are needed to clarify why we cannot detect the signals after salt stress.
Cellular localization of the OsCDPK7 protein was facilitated by the use of isoform-specific antibodies. Previous Western analyses demonstrated that our affinity-purified antibodies recognize a single polypeptide in soluble fractions of shoots and roots of rice plants (Saijo et al. 2000). In addition, they did not cross-react with another recombinant CDPK isoform, OsCDPK2 (Breviario et al. 1995) (data not shown). Immunolocalization with the anti-OsCDPK7 antibodies revealed that the OsCDPK7 protein is expressed most strongly in the vascular bundles of crowns, this expression pattern being essentially the same as that of the mRNA, under cold stress conditions (Fig. 1C, D). Under salt stress conditions, similar patterns of protein localization were observed (Fig. 1E, F). Higher magnification of large vascular bundles clearly showed that the protein is predominantly localized in xylem parenchyma cells surrounding xylem vessels (Fig. 1G). A weak signal was also detected in phloem parenchyma cells. In longitudinal sections, the expression of the OsCDPK7 protein was shown to be highest along with leaf traces under both stresses (Fig. 1H, I; data not shown). Even in the absence of any stress, the same localization of the protein as above was observed (Fig. 1J, K), confirming the previous finding that the OsCDPK7 protein is expressed at an almost constant level irrespective of the mRNA level (Saijo et al. 2000). In our negative control experiments, antigen-absorbed antibodies did not give any detectable signals.
It is possible that the turnover of the protein is accelerated when OsCDPK7 is activated upon stress, the mRNA level increasing to compensate for the loss (Hirt 1999). The regulation should occur predominantly in tissues where OsCDPK7 is expressed at high levels. Thus, it appears that OsCDPK7 promotes both cold- and salt-stress tolerance predominantly in the same regions of crowns. Furthermore, OsCDPK7 seems to function in many developmental stages during the vegetative growth phase of this organ (Fig. 1).
Expression of OsCDPK7 in rice roots
It is known that roots are the primary sensor of salt/drought stress (Davies and Zhang 1991). To gain more insight into the role of OsCDPK7 in stress adaptation in roots, in situ detection of the transcript and immunolocalization of the protein in wild-type rice plants were performed. In the elongation zone, intense signals were found predominantly in the central cylinder, which is composed of specialized conducting tissues, upon cold stress (Fig. 2A–D). A detailed view of a transverse section revealed that the protein is expressed highly in xylem parenchyma cells around metaxylem II and sclerenchyma cells (Fig. 2E, F). These cells are strengthened by lignified secondary walls, and thus may also serve as supporting tissues. Weak signals were also detected in endodermis cells. In contrast, signals were not apparently detected in the epidermis, exodermis or cortex parenchyma. In our most microtome sections, epidermal cells of soil-grown roots were so damaged that they are hard to be seen, although they remained intact in some sections (e.g. Fig. 2E and Fig. 3C) like those of hydroponically grown roots (K. Ishiyama, S. Kojima, F. Takahashi, T. Hayakawa and T. Yamaya, manuscript in preparation). A similar expression pattern as above was observed under salt stress (Fig. 2G, H). Again, a similar expression pattern as above was observed under the normal growth conditions (Fig. 2I, J). Moreover, it should be noted that high-level expression of the OsCDPK7 protein was observed in the root meristem and plerome under all the conditions examined (Fig. 2K, L, data not shown). The signals were also detected in the lateral root primordia (Fig. 2I, J) and the crown root primordia (data not shown, see Fig. 3A), which are considered to be similar to the root meristem.
Localization of the OsCDPK7 pathway in stress-tolerant over-expressing transformants
We were curious to know the mechanisms by which the OsCDPK7 over-expressing plants achieved cold- and salt/drought-stress tolerance. In wild-type plants the abundance of OsCDPK7 protein does not follow the increase in that of its mRNA under these stress conditions, whereas the protein expression at a higher-than-normal level occurs in the transgenic plants with the CaMV 35S promoter (Saijo et al. 2000). The fact prompted us to examine whether or not ectopic expression of OsCDPK7 and ectopic operation of its signaling pathways led to the enhancement in stress tolerance.
For that purpose, we first determined the cellular localization of the OsCDPK7 protein immunohistochemically. Importantly, in the over-expressing transformants under salt-stress conditions, the protein was not ubiquitously expressed but accumulated in essentially the same regions of the crowns and roots as those in wild-type plants (Fig. 3A–D). Similar results were obtained under the normal growth conditions (Fig. 3E–H) and cold-stress conditions (data not shown). Since we performed incubation with antibodies and color development of these sections identically in parallel with the corresponding sections of the wild type, it is clear that the localized protein level was higher in the transformants than in the wild type (compare Fig. 3A and E to Fig. 1E and J; and Fig. 3C and G to Fig. 2G and I). The control antigen-preabsorbed antibodies gave no significant signals.
Next, we carried out in situ hybridization to localize the expression of rab16A, one of the proposed target genes regulated by the salt/drought-stress OsCDPK7 signaling pathway (Saijo et al. 2000), in the OsCDPK7 over-expressing plants. Under salt stress, signals for the transcript were detected most strongly in vascular tissues of both crowns and roots of the transformants (Fig. 3I–L). High-level expression of rab16A was also detected in the meristems of root tips (data not shown). Similar results were obtained for wild-type rice plants exposed to high salinity (data not shown). The rab16A mRNA was not detectable in either the transformants or the wild type under the normal growth conditions (data not shown; Saijo et al. 2000). These results are in good agreement with those for the activity of the rab16A promoter in rice plants (Ono et al. 1996). The cellular localization patterns of the rab16A transcript almost completely coincided with those of the OsCDPK7 protein (Fig. 3). Thus, it seems likely that the OsCDPK7 pathway is amplified preferentially in the properly operating regions in the over-expressing transformants.
Discussion
The present results strongly suggest that the primary physiological function of OsCDPK7 is to protect vascular tissues and the root meristems, both of which are regions susceptible to abiotic stresses. In line with this, the expression of a putative target gene, rab16A, encoding a highly hydrophilic protein that could be involved in water retention, appears to be enhanced by this CDPK pathway predominantly in these regions upon salt stress. Therefore, it is intriguing to presume that the OsCDPK7 pathway plays a role in maintaining osmotic homeostasis in these stress-sensitive regions, at least in part, through the up-regulation of gene expression for LEA proteins.
The observed broad substrate specificity (Saijo et al. 2000) raises a possibility that OsCDPK7 may regulate multiple target proteins. In addition to those directly involved in the machinery of gene expression, a variety of transport proteins, e.g. aquaporins, ion channels and H+-ATPases, which are responsible for cytosolic osmo-regulation can be considered as its substrates. Indeed, some of these proteins have been shown to be altered in their activities by CDPK-mediated phosphorylation (Harmon et al. 2000, Sanders et al. 1999). The identification of in vivo substrate(s), which should be expressed in the above tissues, of OsCDPK7 will further elucidate the mechanism by which this CDPK confers stress tolerance.
It appears that OsCDPK7 functions in essentially the same tissues under both cold- and salt/drought-stress conditions. Although it was suggested that distinct pathways are activated by OsCDPK7 to enhance the cold and salt/drought tolerance (Saijo et al. 2000), it seems very unlikely that these pathways operate in different tissues to maintain the signaling specificity of each pathway. Since the protein levels and tissue localization of OsDCPK7 were unchanged under all the conditions examined, changes in the activity, intracellular location, and/or the availability of a substrate of the enzyme could occur. In this regard, it is noteworthy that Western blot analyses found the presence of a certain amount of the OsCDPK7 protein in the microsomal fractions (105×g pellet) of plant tissues (unpublished results). It is of great interest to determine whether OsCDPK7 is involved in the cold-responsive CDPK activity in the membrane fractions from rice plants (Martin and Busconi 2001).
What is the key for producing stress-tolerant rice plants through over-expression of OsCDPK7 (Saijo et al. 2000)? The current study showed similar OsCDPK7 localization patterns in the transformants to those in wild type with much stronger signals. At present, we cannot exclude a possibility completely that low levels of ectopic expression of the enzyme triggered the stress adaptation processes in the transformants. Another possibility cannot be ruled out either that OsCDPK7 protein is degraded rapidly in tissues other than vascular bundles. Interestingly, however, it has been reported that the activity of the CaMV 35S promoter is particularly high in vascular bundles of both roots and leaves of rice (Terada and Shimamoto 1990). It seems likely that, at least in part, the use of the CaMV 35S promoter in rice fortuitously enabled high-level mRNA accumulation and thus strong protein expression in vascular tissues, where OsCDPK7 is inherently expressed. We propose that not ectopic but proper expression of the protein at a high level gives rise to the increase in stress tolerance. To make use of signaling regulators like CDPKs in plant gene engineering in the future, cellular localization should be taken into consideration.
Materials and Methods
Plant material and growth conditions
Sterilized rice (Oryza sativa L. cv. Notohikari) seeds were germinated, and then transplanted in soil as described (Saijo et al. 2000). The wild-type plants and T2 homozygous OsCDPK7 over-expressing plants (S1) (Saijo et al. 2000) were used. Crowns and roots were obtained from 2-week-old seedlings grown in a growth chamber (16 h light/8 h darkness, 28°C), and 6-week-old plants grown in a greenhouse (16 h light/8 h darkness, 30°C), respectively. Stress treatments were performed before harvest for the indicated periods as described previously (Saijo et al. 2000).
In situ hybridization
Plant tissues were fixed in a solution comprising 4% paraformaldehyde and 0.2% glutaraldehyde (v/v) in 100 mM sodium cacodylate buffer (pH 7.4) for at least 48 h at 4°C. The fixed tissues were dehydrated through a graded series of cold ethanol and n-butanol, and then embedded in paraffin. Microtome sections (10 µm thick) were hybridized with digoxigenin-labeled antisense or sense RNA probes prepared from DNA fragments of OsCDPK7 (nt 1518–2126) (Saijo et al. 2000) and rab16A (nt 669–899) (Yamaguchi-Shinozaki et al. 1989). Hybridization was performed as described previously (Kouchi and Hata 1993) except that the sections were incubated overnight at 52°C.
Immunohistochemical analyses
Microtome sections were prepared as described above. Immunohistochemical analyses were carried out essentially as described previously (Hayakawa et al. 1994). The sections were incubated with affinity-purified anti-OsCDPK7 antibodies (Saijo et al. 2000) diluted 1 : 200, for roots, or 1 : 400, for crowns, in phosphate-buffered saline containing 0.3% (w/v) bovine serum albumin. As negative controls, sections were incubated with the anti-OsCDPK7 antibodies preabsorbed with an excess amount of the antigen.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research on the Priority Areas (No. 12025214) from the Ministry of Education, Science, Sports and Culture of Japan, and the program ‘Research for the Future’ of the Japan Society for the Promotion of Science (JSPS) (JSPS-RFTF96L00604). Y.S. was supported by a Research Fellowship of the JSPS for Young Scientists.
These authors contributed equally to this work.
Present address: Department of Molecular, Cellular, and Developmental Biology, Yale University, 165 Prospect Street, OML 352, New Haven, CT 06520-8104, U.S.A.
Present address: Institute of Biological Chemistry, Clark Hall 299, Washington State University, Pullman, WA 99164-6340, U.S.A.
Corresponding author: E-mail, izui@kais.kyoto-u.ac.jp; Fax, +81-75-753-6470.
Abbreviations
References
Breviario, D., Morello, L. and Giani, S. (
Danyluk, J., Perron, A., Houde, M., Limin, A., Fowler, B., Benhamou, N. and Sarhan, F. (
Davies, W.J. and Zhang, J. (
Garcia, A.B., Engler, J.d., Claes, B., Villarroel, R., Van Montagu, M., Gerats, T. and Caplan, A. (
Harmon, A.C., Gribskov, M. and Harper, J.F. (
Hayakawa, T., Nakamura, T., Hattori, F., Mae, T., Ojima, K. and Yamaya, T. (
Hirt, H. (
Hong, Y., Takano, M., Liu, C.-M., Gasch, A., Chye, M.-L. and Chua, N.-H. (
Houde, M., Daniel, C., Lachapelle, M., Allard, F., Laliberte, S. and Sarhan, F. (
Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O. and Thomashow, M.F. (
Kiegle, E., Moore, C.A., Haseloff, J., Tester, M.A. and Knight, M.R. (
Kouchi, H. and Hata, S. (
Kovtun, Y., Chiu, W.-L., Tena, G. and Sheen, J. (
Lee, J.Y., Yoo, B.C. and Harmon, A.C. (
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (
Martin, M.L. and Busconi, L. (
Ono, A., Izawa, T., Chua, N.-H. and Shimamoto, K. (
Pearce, R.S., Houlston, C.E., Atherton, K.M., Rixon, J.E., Harrison, P., Hughes, M.A. and Dunn, M.A. (
Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K. and Izui, K. (
Sanders, D., Brownlee, C. and Harper, J.F. (
Terada, R. and Shimamoto, K. (