Phylogenetic analysis and subcellular localization of OsHsfC1b
BLAST search and multiple sequence alignment of OsHsfC1b (Os01g53220) revealed homologous proteins in other monocots such as maize, sorghum and Brachypodium
, but also in the dicot Arabidopsis
(Fig. A). In rice, OsHsfC1b shares highest similarity with OsHsfC1a, another member of the four class C HSFs identified in rice (Guo et al. 2008
). All proteins contain a well-conserved N-terminal DNA-binding domain consisting of three α-helices and four β-sheets, and a highly conserved oligomerization domain, also known as HR-A/B domain. In addition, a putative nuclear localization signal (NLS) upstream of the oligomerization domain was identified in all proteins. To confirm targeting of OsHsfC1b to the nucleus, we performed a subcellular localization study, in Arabidopsis
mesophyll cell protoplasts (Fig. B). The fluorescence signal of the GFP–OsHsfC1b fusion protein was detectable mainly in the nucleus and to a lesser extent in the cytosol, as expected for a nuclear protein; the signal of the GFP control was equally distributed over both compartments.
Fig. 1 Phylogenetic analysis and subcellular localization of OsHsfC1b. (A) Multiple sequence alignment of OsHsfC1b and homologous proteins. The DNA-binding domain consists of three α-helices (a1–a3) and four β-sheets (b1–b4). (more ...)
Expression profile of OsHsfC1b in rice roots and leaves exposed to salt, mannitol, ABA or H2O2
We examined the expression of OsHsfC1b in roots and leaves of 4-week-old hydroponically grown rice plants (cv. Nipponbare) exposed to 100 mM NaCl, 100 mM mannitol, 5 µM ABA or 5 mM H2O2 for 30 min or 3 h (Fig. A and B). OsHsfC1b was significantly induced in roots after 30 min treatment with salt, mannitol and ABA. In addition, OsHsfC1b was also significantly upregulated in leaves after 30 min of salt treatment. After 3 h, the expression level of OsHsfC1b in roots was significantly increased by salt and ABA, but not by mannitol. Again, salt stress resulted in an upregulation of expression in leaves. Remarkably, the ABA-triggered induction in roots was ~2-fold higher than that triggered by salt, reaching an ~43-fold and ~33-fold induction after 30 min and 3 h ABA treatment, respectively, as compared with non-stress conditions. H2O2 had no effect on OsHsfC1b transcript level.
Fig. 2 Expression pattern of OsHsfC1a and OsHsfC1b under different treatments. (A) Relative expression of OsHsfC1a and OsHsfC1b in Nipponbare roots treated with 100 mM NaCl, 5 µM ABA, 100 mM mannitol or 5 mM H2O2 for 30 min or 3 h, respectively. (B) (more ...)
In addition to Nipponbare plants, we tested the salt-dependent expression of OsHsfC1b in rice plants of the Dongjin cultivar (Fig. C). In contrast to Nipponbare plants, OsHsfC1b was only induced in roots after 30 min of salt stress, showing an upregulation by ~3.5-fold. We compared the expression profile of OsHsfC1b under the different treatments with the paralogous gene OsHsfC1a (Fig. ). As observed for OsHsfC1b, OsHsfC1a was upregulated in Nipponbare roots exposed to salt stress for 30 min and 3 h, and to ABA treatment for 3 h (Fig. A). Unlike its counterpart, however, OsHsfC1a was not induced by salt stress in Nipponbare leaves (Fig. B). Moreover, it was downregulated by mannitol treatment in roots and induced by ABA in leaves. As shown for OsHsfC1b, OsHsfC1a was significantly induced by salt stress in Dongjin roots (Fig. B).
Identification of the T-DNA insertion line hsfc1b and establishment of amiRNA lines
For functional characterization of OsHsfC1b
, we identified a homozygous T-DNA insertion line (1B-09127.R) in the Dongjin background and named it hsfc1b
. The insertion site is located in the second exon of OsHsfC1b
(Fig. D). Additionally, we generated ami
RNA lines in the Nipponbare background and selected two independent lines, ami
-7.1 and ami
-13.3, for further characterization. Transgenic plants of the T4
) and T1
RNA lines) were analysed regarding the expression of OsHsfC1b
under non-stress conditions. In hsfc1b
roots, we observed an 11-fold reduction of OsHsfC1b
expression, while in ami
-13.3 roots the expression of OsHsfC1b
was decreased by ~5-fold as compared with control plants (Fig. E). The transcript of OsHsfC1b
was not detectable in roots of the ami
-7.1 line. Notably, during salt stress OsHsfC1a
expression in the insertion line was similar to that in the wild type, suggesting that OsHsfC1b
act independently during the stress response [see Additional Information—File 2
Growth of hsfc1b and amiRNA lines under control conditions
Transgenic plants (hsfc1b, ami-7.1 and ami-13.3 lines) showed stunted growth under non-stress conditions, visible 7 days after sowing (DAS) and at the age of 3 weeks (Fig. A and D). Root length and shoot height of seedlings germinated on MS medium were measured 4 and 7 DAS (Fig. B). Shoot length of the hsfc1b mutant was ~75 and ~80 % of that of the wild type (Dongjin) at 4 and 7 DAS, respectively (Fig. B). Furthermore, at 7 DAS, root length of hsfc1b was ~85 % of that of the Dongjin wild type, whereas at 4 DAS no difference was observed between roots of the insertion line and the wild type. The growth retardation was also observed in the ami-7.1 and ami-13.3 lines established in the Nipponbare background (Fig. B). At 4 DAS, both lines had significantly shorter root and shoot lengths than control plants containing the empty vector. Furthermore, at 7 DAS ami-7.1 plants displayed a significantly shorter shoot length, and ami-13.3 plants showed a significantly shorter root length. Besides this, we observed differences in biomass accumulation between hsfc1b and Dongjin wild-type plants (Fig. C). Both shoot fresh weight (FW) and dry weight (DW) of 4-week-old hsfc1b plants were reduced by one-third, and root FW and DW were ~60 % of that of wild-type plants. A similar observation was made for ami-13.3 and ami-7.1 lines, where shoot and root FW and DW were reduced by ~50 and ~75 %, respectively, suggesting that OsHsfC1b functions as a positive regulator of vegetative growth.
Fig. 3 Impact of OsHsfC1b on vegetative growth under normal conditions. (A) Seven-day-old seedlings grown on MS medium. From left to right: Dongjin wild type, hsfc1b, empty-vector control line (Nipponbare background), ami-13.3 line, ami-7.1 line. (B) Root and (more ...)
Growth of hsfc1b, ami-7.1 and ami-13.3 lines under salt stress, osmotic stress or ABA treatment
We examined the salt tolerance of hsfc1b plants. Seeds of the insertion line and the Dongjin wild type were germinated in the presence of either 50 or 100 mM NaCl, and subsequently shoot and root length were determined at 4 and 7 DAS. Under mild stress, shoot length of hsfc1b was significantly more reduced than that of the wild type at both time points (Fig. A). At 100 mM NaCl, a stronger reduction of both shoot and root length as compared with the stressed wild type was observed. These results suggest a requirement of OsHsfC1b for the response to both mild and severe salt stress. For the ami-7.1 line, we consistently observed a 20 % reduction of shoot length at 100 mM NaCl (4 DAS) as compared with stressed empty-vector control plants (Fig. B), while line ami-13.3 did not differ largely from the empty-vector control with respect to shoot and root growth at both time points. We also tested the response of the transgenic lines after 3 weeks of growth in hydroponic culture and subsequent exposure to 50 mM NaCl for 8 days. The hsfc1b insertion line accumulated significantly less FW and DW (shoot and root) as compared with the stressed wild type (Fig. C). Likewise, ami-7.1 and ami-13.3 plants showed a significantly stronger reduction of FW and DW of both shoot and root than empty-vector control plants (Fig. C). Interestingly, Dongjin wild-type and empty-vector control plants (Nipponbare background) also differed regarding their salt tolerance. Whereas the shoot FW and DW were similarly reduced under stress conditions, empty-vector Nipponbare plants were more strongly affected in root FW and DW than Dongjin wild-type plants (Fig. C).
Fig. 4 Impact of OsHsfC1b on growth under stress conditions. (A) Root and shoot length of hsfc1b and Dongjin wild-type seedlings at 4 and 7 DAS grown on MS medium containing 50 or 100 mM NaCl, respectively, relative to non-stressed seedlings. (B) Root and shoot (more ...)
Osmotic stress alone or in combination with salt or drought stress leads to diminished cell growth (Munns and Tester 2008
expression is induced in Nipponbare roots exposed to mannitol (Fig. A). For this reason, we tested the tolerance of hsfc1b
plants to osmotic stress (Fig. D). At 4 DAS, hsfc1b
plants grown on MS medium containing 100 mM mannitol exhibited significantly greater reduced shoot and root lengths, and at 7 DAS a significantly greater reduced root length as compared with the stressed wild type was observed, suggesting a role of OsHsfC1b in the response to osmotic stress (Fig. D).
Abscisic acid is involved in the response to many abiotic and biotic stresses, and exogenous ABA mimics effects caused by environmental stresses (Zhu 2002
). The expression of OsHsfC1b
is ABA-inducible (Fig. A), suggesting a potential role in ABA signalling and/or response. Seedlings of hsfc1b
showed hypersensitivity towards ABA (1 and 5 µM), visible by diminished shoot and root length at 7 DAS (Fig. E); this hypersensitivity was more prominent at 5 µM ABA. Seedlings of ami
-7.1 and ami
-13.3 had major problems in growing on 5 µM ABA, making a quantitative analysis impossible (data not shown). Therefore, we examined these parameters at 1 µM ABA (Fig. F). Whereas the ami
-7.1 and the empty-vector control line showed a similar reduction of shoot and root length, those of ami
-13.3 were significantly more affected (Fig. F).
Expression profiling of genes related to the salt stress response
The data described above indicated that OsHsfC1b contributes to the response to salt and osmotic stress. Next, we wanted to know whether genes known to be salt-responsive in the wild type are affected by the knock-down of OsHsfC1b. We therefore tested the expression of 80 salt-responsive genes involved in salt signalling and ion homeostasis in rice; these genes respond within 24 h of salt stress, with different induction time points and courses (R. Schmidt, MPIMP, Golm, Germany, unpubl. res.). The transgenic plants (hsfc1b and line ami-13.3) were exposed to 100 mM NaCl for 30 min or 3 h, and gene expression was analysed by qRT-PCR. We selected the ami-13.3 line since the reduction in plant size and weight is comparable to that of the T-DNA insertion line (Fig. ). Interestingly, under control conditions, i.e. in the absence of stress, various salt-responsive genes were already differentially expressed in hsfc1b and ami-13.3 lines, compared with the controls (Table ). In hsfc1b roots, we found a significant upregulation of MAP2K.6, the ATPases ECA1, AHA1 and AHA2 as well as VHA-c4, the cation transporters HKT7 and HKT8, and GLR2.8. Additionally, GLR2.7 and TIP2-1 showed a significant downregulation as compared with Dongjin roots. The differentially expressed genes in hsfc1b (Dongjin background) under control conditions differed from those of ami-13.3 (Nipponbare background), possibly indicating cultivar differences. Here the six genes with a change in expression encode MAP3K.4, MAP3K.18, calcineurin-B-like protein CBL7, CAMK1, HAK4 and a protein kinase (Os06g43030) (Table ). Remarkably, the expression of MAPK3K.18 under non-stress conditions was drastically reduced in ami-13.3, showing a >130-fold lower expression than in roots of plants transformed with the empty vector.
Table 1 Expression of genes encoding signalling and ion homeostasis components in hsfc1b and ami-13.3 lines. Comparison of expression levels (log2FC) in roots of hsfc1b, ami-13.3 and their respective controls under non-stress and salt stress conditions (100 mM (more ...)
After 30 min of salt stress, 13 genes were differentially expressed in Dongjin, six of which matched genes that were also differentially expressed in hsfc1b compared with the wild type under control conditions, including ECA1, AHA1, VHA-c4, HKT8, GLR2.7 and TIP2-1, suggesting that the salt stress-associated gene regulatory network (GRN) is already in part activated in hsfc1b even in the absence of salt stress. A similar observation was made when ami-13.3 was compared with empty-vector control plants. After 30 min of salt stress, 15 genes responded in empty-vector control plants including four genes, i.e. MAP3K.4, MAP3K.18, CBL7 and HAK4, which were differentially expressed in ami-13.3 under control conditions (Table ). Exposure of hsfc1b roots to salt stress for 30 min induced nine genes which did not overlap with the genes responding in Dongjin wild-type roots at this time point. After 3 h of salt stress, seven and 13 genes were differentially expressed in Dongjin and hsfc1b roots, respectively. The only overlapping gene at this time point was TIP3-2; five other genes responding in hsfc1b under salt stress overlapped with the 30-min time point for wild-type plants (MAP3K.23, CBL7, HKT8, CaCA/Os11g01580 and TIP2-1). Furthermore, CNGC2 (Os03g55100) showed a strong induction in Dongjin roots after 3 h, but was already induced in hsfc1b after 30 min of salt stress. For ami-13.3, we observed diverging expression profiles at both time points of salt stress as compared with the empty-vector control. After 30 min of salt stress, nine genes were differentially expressed in ami-13.3 roots, of which three genes, i.e. AHA1, HKT8 and CaCA (Os12g42910), showed a similar response in the empty-vector control plants. Similarly, after 3 h of salt stress, we found 16 and 21 genes to be differentially expressed in ami-13.3 and empty-vector control, respectively. Eight genes shared a similar behaviour, including e.g. MPK15, MAP3K.15, CIPK4, TIP2-1 and TIP4-2 (Table ). Overall, these results suggest a temporal misregulation of the expressional network in hsfc1b and ami-13.3 lines.
Irrespective of the duration of salt stress, we observed only a small overlap between the salt stress-associated GRNs of hsfc1b, ami-13.3, Dongjin wild type and the Nipponbare empty-vector line (Fig. A). Of the 19 and 17 genes that responded to salt stress in hsfc1b and Dongjin wild type, respectively, seven genes were in common. Similarly, of the 21 and 29 genes affected by salt stress in the ami-13.3 and empty-vector control lines, respectively, 10 genes showed a similar response. However, when comparing Dongjin wild-type and Nipponbare empty-vector control plants, an overlap of only seven salt-responsive genes was observed, of which four genes showed contrasting responses (Table ), suggesting divergent expressional responses of Dongjin and Nipponbare to salt stress. Furthermore, 29 genes responded to salt stress in Nipponbare control roots, which is almost twice the number of genes differentially expressed in Dongjin wild-type roots.
Fig. 5 Expressional response of salt stress-responsive genes in the different genotypes. (A) Venn-diagram presentation of the overlap of differentially expressed genes in hsfc1b, ami-13.3 and corresponding controls under salt stress. (B) Differentially expressed (more ...)
Expression profiling of sHSP genes
Recently, 12 sHSP
genes were found to respond to salt stress in rice (Sarkar et al. 2009
). Since HSFs potentially regulate sHSP
gene expression under stress conditions, we tested the expression of these 12 genes in ami
-13.3 roots (Fig. B). In the absence of salt stress, Hsp23.6-MII
was already induced in ami
-13.3 compared to the Nipponbare empty-vector line. After 30 min of salt stress, Hsp18.1-CII
were significantly induced in empty-vector roots, whereas Hsp18.6-CIII
was downregulated. In addition to Hsp23.2-ER
also showed an increased expression in ami
-13.3 roots under salt stress. Like in empty-vector control plants, Hsp18.6-CIII
was downregulated in ami
-13.3 under salt stress, whereas Hsp18.1-CII
was not significantly affected. For Hsp18.6-CIII
, we observed a stronger response in ami
-13.3 under salt stress than in empty-vector control roots. There was no significant change in the expression of Hsp16.0-px
-13.3 or empty-vector control roots under salt stress.