Os12g0597000

CBL proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer.

Annotated Information

Function

Table1 Amino acid similarity and identity of rice CBLs (OsCBL1–10) and Arabidopsis CBLs (AtCBL1–10). For each pairwise comparison, similarity values are followed by identity values in parentheses.(from reference)

Figure 1 Yeast two-hybrid analysis demonstrates an interaction between OsCBLs and AtCIPKs. OsCBLs and AtCIPKs were translationally fused to the GAL4 DNA-binding domain (BD) and activation domain (AD) as indicated. Nutritional reporter systems minus Leu plus Trp (−LT) and minus Leu, Trp, and His (−LHT) and filter-lift GAL assays were employed to examine the interaction between OsCBLs and AtCIPKs (A). A positive control showing the interaction of AtCBL1 with AtCIPK1 is shown in B.(from reference)

Figure 2 OsCBL2 to 4 are localized to membranes. OsCBL1 to 4 were translationally fused to GFP and transiently expressed in barley aleurone protoplasts. The figure shows representative epifluorescence images (top) and bright-field images (bottom) of single, transformed cells. The unmagnified width of each image is approximately 40 μm.(from reference)

Figure 3 Antisense OsCBL2 or HvCBL2delays the GA-induced vacuolation of barley aleurone protoplasts. Barley protoplasts were cotransfected with GFP andAsOsCBL2, GFP, andAsHvCBL2, or with GFP and empty cassette (pLZUbi) using the constructs diagrammed in A. The extent of vacuolation for individual protoplasts was scored using the five categories indicated in B. Vacuoles are seen as dark regions surrounded by bright regions of cytoplasm. The number of protoplasts in each category 48 h after transfection and 42 h after treatment with GA are shown in C forAsOsCBL2 and in D for AsHvCBL2.(from reference)

Figure 4 Antisense OsCBL2 does not delay GA-induced transcription of GUS from anα-amylase promoter in rice half-grain. A diagram of the constructs introduced by particle bombardment is shown in A. Transcription of GUS from a GA-regulated α-amylase promoter was measured relative to expression of LUX (GUS:LUX ratio) driven by a constitutive ubiquitin promoter (B). Half-grains were incubated for 24 h without hormone (−GA) or with GA and the ratio of GUS-to-LUX expression determined in the presence and absence of the antisense construct.(from reference)

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer.

OsCBLs Interact with AtCIPKs

We used the yeast two-hybrid system to demonstrate that rice CBLs interact with AtCIPKs. OsCBL1 to 4 were fused to the binding domain of GAL4, whereasAtCIPK1, 6, and 8 were fused to the activation domain of GAL4. Figure 1A shows the growth of yeast on selection medium and the corresponding assay for β-galactosidase when these different OsCBLs and AtCIPKs were used as bait and prey. As expected, the positive control showed interaction between AtCBL1 and AtCIPK1 (Fig. 1B). OsCBL2, which has 74% amino acid similarity with AtCBL1 (Table I), also had a strong interaction with AtCIPK1. Like AtCBL1, OsCBL2 interacted strongly with AtCIPK8 and weakly with AtCIPK6. OsCBL4 also interacted strongly with AtCIPK1 and 8, but unlike OsCBL2, it did not interact with AtCIPK6. OsCBL1 and 3 both interacted with all three of the Arabidopsis CIPKs examined. These data provide evidence that OsCBL1 to 4 proteins are functional homologs of Arabidopsis CBL proteins.

Specificity for rice CBL function is likely to arise from differences in intracellular localization and different timing of expression. We show here that OsCBL2 and 3are targeted to the TN, and OsCBL4 to the PM (Fig. 2). Even though both OsCBL2 and 3 are targeted to the TN, their roles may be distinguished by the timing of their expression. For example, OsCBL2 is expressed in aleurone during germination, but OsCBL3 was not detectable in this tissue under the conditions that we have tested. OsCBL2 may be involved in vacuole function since transformation of aleurone protoplasts with an antisense construct of OsCBL2 orHvCBL2 slowed the rate of GA-induced vacuolation (Fig. 3), but not GA-induced transcription of an α-amylase reporter construct (Fig. 4).

Expression

OsCBL2 high expression in booting culms, young spikes, seedling roots and shoots. Expression of OsCBL2 is not induced by salt, drought, cold or ABA treatment. Although both OsCBL1 and 2 were expressed in rice half-grains, OsCBL2 was specifically up-regulated by GA (Fig. 5). GeneChip and RNA blotting experiments showed that OsCBL2 was most strongly expressed in aleurone and root and, using an expression intensity value of 50 as a cutoff, it is clear thatOsCBL2 is expressed in most tissues of the rice plant.

In aleurone cells, GA stimulates the synthesis and secretion of hydrolytic enzymes including α-amylase, promotes the vacuolation of the aleurone protoplast, and initiates programmed cell death. All of these processes require an increase in [Ca2+]cyt. Here we show that the expression of one gene in the rice CBL family is up-regulated in aleurone by GA, but not by ABA. We show that other rice CBLs are not differentially expressed by GA and ABA in aleurone or in vegetative tissues of the shoot or root. We present data showing that OsCBL2 is localized to the aleurone tonoplast (TN), and transient expression assays with rice and barley CBLs in barley aleurone cells indicate that they are likely to be involved in a GA-signaling pathway that leads to the vacuolation of the aleurone cell.

Hormone and Tissue-Specific Expression of OsCBLs

Only OsCBL2 contains the probe sequences found on the rice GeneChip microarray. It is therefore highly likely that the GA-regulated CBL identified in our microarray experiments (Fig. 6) is OsCBL2. We used the GeneChip microarray to quantitate the expression of OsCBL2 in the tissues of rice cv Nipponbare at all stages of development. These data are presented in Figure 7, where GeneChip intensity values for each tissue or organ are plotted with higher values farther from the center of the figure. OsCBL2 is expressed at high levels in roots of seedlings and tillering plants, during early stages of panicle and seed formation, and in the aleurone of mature grain. Expression of OsCBL2 was lowest in mature leaves and stems and in the emerging inflorescence shoot (Fig. 7).

To investigate the expression of OsCBLs in germinating Nipponbare rice seedling tissues, RNA was isolated from scutellum, shoots, and roots of 7-d-old seedlings and northern blots were hybridized with gene-specific probes for OsCBL1 to 3(Fig. 8). OsCBL2 is expressed in all rice seedling tissues and this confirmed the analysis made with the GeneChip array (Fig. 7). RNA blotting also confirmed thatOsCBL2 mRNA was abundant in roots relative to shoots and scutella, whereas theOsCBL1 transcript was more abundant in shoots than in roots and the OsCBL3transcript was abundant in both root and shoot tissue (Fig. 8). OsCBL4 and 7 were not expressed strongly enough in tissues of 7-d-old seedlings to be detected.

GA-Induced Expression of OsCBL2 Is Reduced in the Aleurone Layer of dwarf1 Mutant Rice

We also used RNA profiling and northern blotting to see whether GA-induced expression of OsCBL2 in aleurone cells was dependent on a signaling pathway that utilizes heterotrimeric G-proteins. For these experiments, RNA was isolated from half-grains of wild-type and dwarf1 (d1) mutant rice. The d1 rice mutant lacks the α-subunit of heterotrimeric G-proteins and shows a defective GA response, except at high GA concentrations. In the experiment shown in Figure 9A, there was a 3-fold increase in OsCBL2 expression in wild-type rice aleurone after 8-h incubation at a high (5 μM) GA concentration. When wild-type half-grains were incubated with a low (100 nM) GA concentration,OSCBL2 expression was still almost twice as high as that at time zero (Fig. 9A). Expression of OsCBL2 in d1 half-grains, however, was much reduced at 5 μM GA compared to wild type, and transcript abundance was virtually unchanged following 8-h incubation with 100 nM GA (Fig. 9A). Similar changes in expression were observed for α-amylase in d1 and wild-type rice half-grains (Fig. 9B). Thus, there was virtually no change in the expression of the RAmy1A gene at low GA concentrations in d1 rice, whereas in wild-type rice grain low GA brought about a large change in RAmy1A expression (Fig. 9B). RNA blotting was used to confirm the microarray data on CBL expression as shown in Figure 9C. Expression ofOsCBL2 was observed in wild-type aleurone and the d1 mutant at 5 μM GA, butOsCBL2 transcript could not be detected in the d1 mutant at 100 nM GA.

Mutation

The amount of OsCBL2 transcript was increased specifically by GA treatment in rice aleurone (Figs.5,6, and 9). Using microarray analyses and RNA blots, we show that the up-regulation ofOsCBL2 expression occurs within 3 h of GA treatment and persists for at least 48 h (Figs.5,6, and 9). Data from experiments with the d1 mutant of rice strongly suggest that OsCBL2 transcription is part of a GA-signaling pathway that involves the α-subunit of heterotrimeric G-proteins (Fig. 9).

OsCBL2 expression in aleurone is specifically up-regulated by GA (Figs. 5 and 6). Transcript abundance was unchanged when rice half-grains were incubated with ABA or no hormone, or when seedlings were exposed to various stresses. Perhaps more interesting is our observation that correct expression of OsCBL2 in aleurone protoplasts seems to be required for proper vacuolation (Fig. 3). When barley aleurone protoplasts were transiently transformed with antisense constructs forOsCBL2 or HvCBL2 (Fig. 3, C and D), vacuolation was retarded. This was a specific effect in that AsOsCBL2 did not inhibit transcription from an α-amylase promoter (Fig. 4). One interpretation of these data is that OsCBL2 interacts with one or more proteins in aleurone cells, and that an insufficient amount of OsCBL2 leads to a defect in vacuole function. For example, OsCBL2 may activate a CIPK and the OsCBL2/CIPK complex may promote vacuole fusion and enlargement. AntisenseOsCBL2 would reduce the amount of OsCBL2 and prevent the formation of the active OsCBL/CIPK complex. This speculation is consistent with our previous data showing that a Ser/Thr protein kinase present on the TN in barley aleurone protoplasts is involved in the gating of a Ca2+-regulated ion channel.

Knowledge Extension

A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Calcium-binding proteins with similarity to calcineurin B have been cloned recently from plants . These calcineurin B-like proteins (CBLs) contain calcium-binding EF hands and are similar to the regulatory B-subunit of calcineurin and to the neuronal calcium sensor . CBLs, therefore, have the potential to transduce [Ca2+]cyt signals and are thought to play roles in stress and hormone signaling in plants . The first CBL gene to be cloned was a salt overly sensitive (SOS) gene from Arabidopsis (Arabidopsis thaliana) that was designatedSOS3 . SOS3 is identical to AtCLB4, a salt-responsive CBL gene cloned independently from Arabidopsis . At least 10 expressed CBL genes and proteins from Arabidopsis have now been identified, and many CBL genes are present in the sequenced rice (Oryza sativa) genome.

Labs working on this gene

Department of Plant and Microbial Biology, University of California, Berkeley, California 94720–3102 (Y.-s.H., P.C.B., Y.H.C., R.L.J.); and Torrey Mesa Research Institute, Syngenta Research and Technology, San Diego, California 92121 (H.-S.C., T.Z.); State key lab of crop genetics and germplasm enhancement, Nanjing Agricultural University, Nanjing, 210095, PR China; College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, 321004, PR China