Accepted Manuscript The CNT1 Domain of Arabidopsis CRY1 alone Is Sufficient to Mediate Blue-light Inhibition of Hypocotyl Elongation Sheng-Bo He, Wen-Xiu Wang, Jing-Yi Zhang, Feng Xu, Hong-Li Lian, Ling Li, HongQuan Yang PII:
S1674-2052(15)00140-9
DOI:
10.1016/j.molp.2015.02.008
Reference:
MOLP 94
To appear in:
MOLECULAR PLANT
Received Date: 12 November 2014 Revised Date:
14 February 2015
Accepted Date: 18 February 2015
Please cite this article as: He S.-B., Wang W.-X., Zhang J.-Y., Xu F., Lian H.-L., Li L., and Yang H.-Q. (2015). The CNT1 Domain of Arabidopsis CRY1 alone Is Sufficient to Mediate Blue-light Inhibition of Hypocotyl Elongation. Mol. Plant. doi: 10.1016/j.molp.2015.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT LETTERS TO THE EDITOR The CNT1 Domain of Arabidopsis CRY1 alone Is Sufficient to Mediate Blue-light
RI PT
Inhibition of Hypocotyl Elongation
Sheng-Bo Hea, Wen-Xiu Wanga, Jing-Yi Zhanga, Feng Xua, Hong-Li Lianb, Ling Lib
Key Laboratory of Urban Agriculture (South) Ministry of Agriculture and School of
Agriculture and Biology; b
M AN U
a
SC
& Hong-Quan Yangb,c,1
School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800
Dongchuan Road, Shanghai 200240, China.
Collaborative Innovation Center of Genetics and Development, Fudan University,
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c
Shanghai 200433, China
To whom correspondence should be addressed.
EP
1
AC C
E-MAIL: [email protected]; FAX (086) 21-3420-5877.
Running title:
Light signaling mediated by CNT1
1
ACCEPTED MANUSCRIPT Dear Editor, Cryptochromes (CRY) are photolyase-like blue light receptors that mediate various light responses in plants and animals. In Arabidopsis, there are two cryptochromes,
CRY1
and
CRY2,
which
mainly
regulate
RI PT
homologous
photomorphogenesis and photoperiodic flowering, respectively (Guo et al., 1998; Lin et al., 1998). Cryptochromes are structurally divided into N-terminal domain related
SC
to photolyase and C-terminal extension domain (Cashmore, 2003; Yang et al., 2000).
M AN U
The C-terminal domain of CRY1 and CRY2 [CCT1 (residues 490-681) and CCT2 (residues 486-612), also known as CCE1 and CCE2] is shown to mediate blue light signaling, whereas the N-terminal domain of CRY1 and CRY2 [CNT1 (residues 1-489) and CNT2 (residues 1-485)] is known to bind chromophore to sense blue light and
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mediate CRY dimerization (Brautigam et al., 2004; Sang et al., 2005; Yang et al., 2000). Specifically, CRY1-mediated blue light signaling involves CCT1-mediated interactions with COP1 and SPA1 (Lian et al., 2011; Liu et al., 2011; Wang et al.,
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2001; Yang et al., 2001). CCT2 also mediates CRY2-COP1 interaction (Wang et al.,
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2001). The functional region of CCT2 is further confined to NC80 motif (residues 486-565), which is proposed to function through similar mechanisms to CCT2 (Yu et al., 2007). Interestingly, CCT2 doesn’t mediate CRY2-SPA1 interaction, which is mediated by CNT2 (Zuo et al., 2011). CRY2 is also found to interact with a family of bHLH transcription factors, cryptochrome-interacting bHLH (CIBs) (Liu et al., 2008), through photolyase homology region (PHR, residues 1-498) (Kennedy et al., 2010), which comprises CNT2 (Sang et al., 2005; Yang et al., 2000) and a part of NC80 (Yu 2
ACCEPTED MANUSCRIPT et al., 2007). However, it remains to be determined whether CNT2 or a part of NC80 mediates CRY2-CIBs interactions. In addition, no direct evidence shows whether CRY2 is able to promote flowering independent of CCT2/NC80-mediated signaling.
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CRY1 and CRY2 sequence alignment reveals a region within CCT1 (residues 489-619) showing similarity to CRY2-NC80, which is here named CRY1-NC80 and doesn’t overlap with CNT1 (Supplemental Figures 1A and 1B). Given that CCT1 and
SC
likely its NC80 are involved in CRY1 signaling, whether CNT1 alone is able to
M AN U
mediate blue-light inhibition of hypocotyl elongation remains unknown. To explore the potential function of CNT1, we sought to make transgenic cry1 mutant lines overexpressing CNT1 and examine their hypocotyl elongation phenotype in blue light. In our previous study, overexpression of CNT1 without the nuclear
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localization signal sequence (NLS) tag in wild type background leads to a dominant negative phenotype under blue light, but fails to confer a phenotype in cry1 mutant background (Sang et al., 2005). Since CRY1 is shown to be localized in both
EP
cytoplasm and nucleus and the nuclear localization is required for CRY1 regulation of
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hypocotyl elongation (Wu and Spalding, 2007), the failure for CNT1 to mediate a light response in cry1 mutant background might result from insufficient nuclear abundance of CNT1. Therefore, we generated transgenic cry1 mutant plants overexpressing CNT1-NLS-YFP (35S::CNT1-NLS-YFP or CNT1; Figure 1A) and the control GUS-NLS-YFP (35S::GUS-NLS-YFP or GUS; Supplemental Figure 2A) fusion protein, respectively. All the GUS lines examined that had strong nuclear YFP signals showed a cry1-like phenotype. Of 80 independent CNT1 lines in T1 examined, 3
ACCEPTED MANUSCRIPT 15 lines without YFP signals exhibited no phenotype in blue light, whereas the remaining 65 lines with nuclear YFP signals displayed varied degrees of shortened hypocotyls, of which 34 lines with the strongest YFP signals hardly elongated, and
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exhibited extremely compact morphology without elongated stem after transferred to soil and eventually died (Supplemental Figure 2B). We failed to obtain homozygotes of even weak CNT1 lines likely due to impaired fertility. Therefore, we selected three
SC
weak heterozygous lines, CNT1#8, #9, and #13, for detailed phenotypic analyses.
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All these three lines segregated three classes of siblings in T2, which showed strong, intermediate, and no hypocotyl phenotype, respectively, at a ratio of about 1:2:1, and the results for CNT1#9 were shown (Figures 1B and 1C). The expression of CNT1 was strictly correlated with short-hypocotyl phenotype in all these three lines
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(Figure 1D). Moreover, the siblings segregated from CNT1#9 that had strongly shortened hypocotyls expressed more CNT1 protein than those that had intermediately shortened hypocotyls, while those that had cry1-like hypocotyls didn’t
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express CNT1 (Figure 1E), indicating that CNT1 inhibits hypocotyl elongation in a
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dose-dependent manner in blue light. Intriguingly, CNT1 failed to confer a phenotype in either darkness or red or far-red light (Figures 1F and 1G and Supplemental Figures 3A-D), indicating that CNT1 inhibits hypocotyl elongation in a blue-light-dependent manner. Besides the hypocotyl phenotype, we also observed multiple developmental defects for CNT1 plants, including reduced plant size and height, decreased silique length, sterility, and increased shoot branching (Supplemental Figures 2B-E). Notably, of the twenty-two missense mutations in hy4/cry1 alleles, thirteen 4
ACCEPTED MANUSCRIPT mutations occur within the N terminus (Ahmad et al., 1995; Shalitin et al., 2003), demonstrating the functional importance of this domain. We generated transgenic cry1 plants overexpressing mutant CNT1-NLS-YFP corresponding to eight missense cry1
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alleles, respectively (Figure 1A). In contrast to CNT1 seedlings, all the T1 transgenic seedlings harboring any one of the eight mutant CNT1 transgenes displayed cry1-like long hypocotyls in blue light. Low levels of mutant CNT1 proteins were uniformly
SC
detected in multiple lines for each one of mutant CNT1 transgenes except for
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CNT1-A462V (corresponding to cry1-305) and CNT1-G283E (corresponding to hy4-5) transgenes. All the representative lines for mutant transgenes exhibited a similar phenotype to cry1 mutant in blue light, of which only CNT1-A462V#4 and CNT1-G283E#3 lines expressed similar levels of CNT1 proteins to CNT1#9 (Figures
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1H-J). We obtained similar results when transiently expressing mutant CNT1s in tobacco leaves (Supplemental Figure 4). Subcellular localization analyses showed that, regardless of the N- or C-terminal YFP fusion, CRY1-A462V and CRY1-G283E
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displayed similar cytoplasmic and nuclear localization patterns to CRY1 in
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Arabidopsis protoplasts or epidermal cells of tobacco leaves (Supplemental Figures 5 and 6), indicating that A462V and G283E mutations likely interfere with CRY1 activity rather than subcellular localization. Taken together, these data indicate that the mutations within CNT1 may affect protein stability and/or properties, thus eliminating its capacity to confer enhanced blue light inhibition of hypocotyl elongation in a manner strikingly similar to the effects of these mutations on the full-length CRY1. To further confirm CNT1 function, we placed YFP at the N terminus of CNT1 5
ACCEPTED MANUSCRIPT and CNT1 mutants (CNT1-A462V and -G283E) and transformed them into cry1 mutant, respectively (Supplemental Figure 7A). We observed that overexpression of YFP-NLS-CNT1 resulted in varied degrees of short-hypocotyl phenotypes in blue
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light, whereas that of CNT1-A462V and CNT1-G283E failed to inhibit hypocotyl elongation (Supplemental Figures 7B-E). These results together with those from CNT1-NLS-YFP overexpression indicate that CNT1 overexpression inhibits
then
performed
RNA-seq
analyses
with
dark-grown
WT,
and
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We
SC
hypocotyl elongation regardless of N- or C-terminal YFP fusion.
blue-light-grown WT, cry1 cry2, CNT1#9, and CNT1-G283E#3 seedlings. The results showed that CRY regulated a large number of blue light-controlled genes in a strikingly similar manner (3,436, designated CRY-regulated genes; Supplemental
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Figure 8 and Supplemental Table 1). Notably, CNT1 influenced about one third (1,151) of CRY-regulated genes (Figure 1K and Supplemental Table 1), and regulated 998 (87%) genes in the same direction as CRY did (Figure 1L). Gene Ontology (GO)
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analyses demonstrated that CNT1 and CRY are highly involved in the regulation of
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multiple overlapping biological processes including cell wall remodeling and growth-related phytohormones signaling (Supplemental Figure 9). These results indicate that CNT1 is involved in mediating CRY1 signaling. We then compared CNT1-regulated genes with the previously-identified genes
responsive to auxin, brassinosteroids (BR), and gibberellins (GA), respectively. The results showed that the majority of overlapping genes were regulated in an opposite direction by CNT1 and each one of these three phytohormones, auxin (83/116, 72%), 6
ACCEPTED MANUSCRIPT BR (356/488, 73%), and GA (86/108, 80%), respectively (Figures 1M-O and Supplemental Tables 2-4), indicating that the antagonistic regulation of hypocotyl elongation by CNT1 and these phytohormones is likely mediated through antagonistic
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regulation of gene expression. Next, we performed qRT-PCR to confirm CRY and CNT1 regulation of genes that are responsive to auxin, BR, and GA by selecting ten representative genes, whose
SC
expression is shown to be up-regulated by at least one of auxin, BR, and GA.
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Moreover, the selected SAUR19-SAUR24 subfamily genes, SAUR63, PRE1, PRE5, EXP8, and HAT2, are shown to promote hypocotyl elongation. We found that the expression of each one of these ten genes was drastically more reduced in WT seedlings grown in blue light than in those grown in darkness, while less inhibited in
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cry1 cry2 or cry1 than in WT in blue light (Figure 1P and Supplemental Figure 10). Strikingly, CNT1 overexpression considerably reduced the expression of these genes, whereas CNT1-G283E or CNT1-A462V overexpression did not (Figure 1P and
at
least
in
part,
through
CNT1-mediated
repression
of
the
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light,
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Supplemental Figure 11), indicating that CRY1 inhibits hypocotyl elongation in blue
auxin/BR/GA-responsive gene expression. CNT1 may confer blue light response by working together with CRY2 to inhibit
COP1-SPA complex through CCT2 and/or by interacting with unidentified factors to mediate pathways that are totally separate from COP1/SPA-mediated pathway. Since CNT1 overexpression in wild type background leads to dominant negative effects on endogenous CRY1 through CNT1-CRY1 interaction (Sang et al., 2005), the enhanced 7
ACCEPTED MANUSCRIPT blue light response conferred by CNT1 in cry1 may not result from the enhanced CRY2 activity by CNT1-CRY2 interaction. Indeed, CNT1 overexpression in cry1 cry2 double mutant resulted in varied degrees of shortened hypocotyls in blue light
enhanced
accumulation
of HY5,
whereas
CNT1
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(Supplemental Figures 12A and 12B). Interestingly, CCT1 overexpression led to overexpression
did
not
(Supplemental Figure 13). These results strongly indicate that CNT1-mediated inhibition
of hypocotyl
elongation
does
not
proceeds
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blue-light
through
(Supplemental Figure 14).
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CCT1/CCT2-COP1/SPA-HY5 pathway, but likely through totally new pathways
We revealed a previously unrecognized function of CNT1 in mediating light responses in Arabidopsis, which does not require CCT1, thus advancing our
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understanding of CRY1 signaling mechanism and opening up a new avenue for exploring CRY1-interacting proteins. Great efforts should be made to characterize the potential CNT1-interacting proteins in future study. Given that Arabidopsis CRY2
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interacts with transcription regulators likely through CNT2 (Kennedy et al., 2010) and
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that type I animal CRY is clearly shown to mediate signaling through its N terminus-mediated interactions with transcription regulators (Busza et al., 2004), as well as that CNT1 is implicated in the inhibition of auxin/BR/GA-responsive gene expression (Figures 1M-P), it will be worth exploring whether CNT1 might directly regulate the transcription regulators in auxin/BR/GA signaling pathways.
FUNDING 8
ACCEPTED MANUSCRIPT This work was supported by grants from the National Natural Science Foundation of China (90917014, 91217307, and 30830012 to H.-Q.Y., and 31170266 to H.-L.L.).
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China Innovative Research Team, Ministry of Education, and 111 Project (B14016).
REFERENCES
Ahmad, M., Lin, C., and Cashmore, A.R. (1995). Mutations throughout an
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Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin
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accumulation and inhibition of hypocotyl elongation. Plant J. 8:653-658. Brautigam, C.A., Smith, B.S., Ma, Z., Palnitkar, M., Tomchick, D.R., Machius, M., and Deisenhofer, J. (2004). Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA.
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101:12142-12147.
Busza, A., Emery-Le, M., Rosbash, M., and Emery, P. (2004). Roles of the two Drosophila
CRYPTOCHROME
structural
domains
in
circadian
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photoreception. Science 304:1503-1506.
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Cashmore, A.R. (2003). Cryptochromes: enabling plants and animals to determine circadian time. Cell 114:537-543.
Guo, H., Yang, H., Mockler, T.C., and Lin, C. (1998). Regulation of flowering time by Arabidopsis photoreceptors. Science 279:1360-1363.
Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D., and Tucker, C.L. (2010). Rapid blue-light-mediated induction of protein interactions in living cells. Nature Methods 7:973-975. 9
ACCEPTED MANUSCRIPT Lian, H.L., He, S.B., Zhang, Y.C., Zhu, D.M., Zhang, J.Y., Jia, K.P., Sun, S.X., Li, L., and Yang, H.Q. (2011). Blue-light-dependent interaction of cryptochrome 1 with
SPA1
defines
a dynamic signaling
mechanism.
Genes
Dev.
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25:1023-1028. Lin, C., Yang, H., Guo, H., Mockler, T., Chen, J., and Cashmore, A.R. (1998). Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light
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receptor cryptochrome 2. Proc. Natl. Acad. Sci. USA. 95:2686-2690.
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Liu, B., Zuo, Z., Liu, H., Liu, X., and Lin, C. (2011). Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev. 25:1029-1034.
Liu, H., Yu, X., Li, K., Klejnot, J., Yang, H., Lisiero, D., and Lin, C. (2008).
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Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322:1535-1539. Sang, Y., Li, Q.H., Rubio, V., Zhang, Y.C., Mao, J., Deng, X.W., and Yang, H.Q.
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(2005). N-terminal domain-mediated homodimerization is required for
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photoreceptor activity of Arabidopsis CRYPTOCHROME 1. Plant Cell 17:1569-1584.
Shalitin, D., Yu, X., Maymon, M., Mockler, T., and Lin, C. (2003). Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis
cryptochrome 1. Plant Cell 15:2421-2429. Wang, H., Ma, L.G., Li, J.M., Zhao, H.Y., and Deng, X.W. (2001). Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. 10
ACCEPTED MANUSCRIPT Science 294:154-158. Wu, G., and Spalding, E.P. (2007). Separate functions for nuclear and cytoplasmic cryptochrome 1 during photomorphogenesis of Arabidopsis seedlings. Proc.
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Natl. Acad. Sci. USA. 104:18813-18818. Yang, H.Q., Tang, R.H., and Cashmore, A.R. (2001). The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1. Plant Cell
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13:2573-2587.
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Yang, H.Q., Wu, Y.J., Tang, R.H., Liu, D., Liu, Y., and Cashmore, A.R. (2000). The C termini of Arabidopsis cryptochromes mediate a constitutive light response. Cell 103:815-827.
Yu, X., Shalitin, D., Liu, X., Maymon, M., Klejnot, J., Yang, H., Lopez, J., Zhao, X.,
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Bendehakkalu, K.T., and Lin, C. (2007). Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2. Proc. Natl. Acad. Sci. USA. 104:7289-7294.
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Zuo, Z., Liu, H., Liu, B., Liu, X., and Lin, C. (2011). Blue light-dependent interaction
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of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis. Curr. Biol. 21:841-847.
FIGURE LEGENDS
Figure 1. CNT1 alone is sufficient to inhibit hypocotyl elongation. (A) Schematic diagram depicting the construct expressing CNT1-NLS-YFP used for transformation of cry1 mutant. Various point mutations are also indicated. NLS indicates nuclear localization signal. 11
ACCEPTED MANUSCRIPT (B, F, and H) Phenotypes of 5-day-old seedlings. All the lines harboring the constructs shown in (A) are in cry1 background. S, M, and L denote short, intermediate, and long hypocotyls of siblings segregated from heterozygous CNT1#9
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line (B), respectively. Dk (F) and BL (B and H) denote darkness and blue light (30 µmol/m2/s for B; 20 µmol/m2/s for H), respectively. Bar=2 mm.
(C and I) Statistical analyses of hypocotyl length corresponding to (B and H),
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respectively. The numbers of segregated seedlings for each phenotype were shown
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above each column (C). Data are means ± SD (n was shown above each column for C; n=30 for I).
(D, E, G, and J) Western blot analysis showing CNT1 protein accumulation. S and L denote samples from blue-light-grown siblings with short and long hypocotyls
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segregated from the indicated heterozygous CNT1 lines, respectively (D). The results shown in (E), (G), and (J) correspond to samples from seedlings shown in (B), (F), and (H), respectively.
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analyses.
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(K) Venn diagram showing overlapping genes among datasets from RNA-seq
(L) Hierarchical clustering analysis of 1,151 overlapping genes as shown in (K). (M-O) Venn diagrams showing the overlapping genes between datasets and hierarchical clustering analyses showing largely converse regulation of overlapping genes by CNT1 and auxin (M) or brassinosteroids (BR, N) or gibberellins (GA, O). The scale bar indicates the log2 value of fold change (L-O). (P) qRT-PCR analysis showing the effects of CNT1 and CNT1-G283E on 12
ACCEPTED MANUSCRIPT auxin/BR/GA-responsive gene expression. Blue-light-grown seedlings with the indicated genotypes were used. The relative expression was normalized to that in WT arbitrarily set to 1. Data are means ± SD (n=2 for IAA5 and SAUR15; n=3 for other
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SC
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genes).
13
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ACCEPTED MANUSCRIPT
S1674-2052(15)00140-9
DOI:
10.1016/j.molp.2015.02.008
Reference:
MOLP 94
To appear in:
MOLECULAR PLANT
Received Date: 12 November 2014 Revised Date:
14 February 2015
Accepted Date: 18 February 2015
Please cite this article as: He S.-B., Wang W.-X., Zhang J.-Y., Xu F., Lian H.-L., Li L., and Yang H.-Q. (2015). The CNT1 Domain of Arabidopsis CRY1 alone Is Sufficient to Mediate Blue-light Inhibition of Hypocotyl Elongation. Mol. Plant. doi: 10.1016/j.molp.2015.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT LETTERS TO THE EDITOR The CNT1 Domain of Arabidopsis CRY1 alone Is Sufficient to Mediate Blue-light
RI PT
Inhibition of Hypocotyl Elongation
Sheng-Bo Hea, Wen-Xiu Wanga, Jing-Yi Zhanga, Feng Xua, Hong-Li Lianb, Ling Lib
Key Laboratory of Urban Agriculture (South) Ministry of Agriculture and School of
Agriculture and Biology; b
M AN U
a
SC
& Hong-Quan Yangb,c,1
School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800
Dongchuan Road, Shanghai 200240, China.
Collaborative Innovation Center of Genetics and Development, Fudan University,
TE D
c
Shanghai 200433, China
To whom correspondence should be addressed.
EP
1
AC C
E-MAIL: [email protected]; FAX (086) 21-3420-5877.
Running title:
Light signaling mediated by CNT1
1
ACCEPTED MANUSCRIPT Dear Editor, Cryptochromes (CRY) are photolyase-like blue light receptors that mediate various light responses in plants and animals. In Arabidopsis, there are two cryptochromes,
CRY1
and
CRY2,
which
mainly
regulate
RI PT
homologous
photomorphogenesis and photoperiodic flowering, respectively (Guo et al., 1998; Lin et al., 1998). Cryptochromes are structurally divided into N-terminal domain related
SC
to photolyase and C-terminal extension domain (Cashmore, 2003; Yang et al., 2000).
M AN U
The C-terminal domain of CRY1 and CRY2 [CCT1 (residues 490-681) and CCT2 (residues 486-612), also known as CCE1 and CCE2] is shown to mediate blue light signaling, whereas the N-terminal domain of CRY1 and CRY2 [CNT1 (residues 1-489) and CNT2 (residues 1-485)] is known to bind chromophore to sense blue light and
TE D
mediate CRY dimerization (Brautigam et al., 2004; Sang et al., 2005; Yang et al., 2000). Specifically, CRY1-mediated blue light signaling involves CCT1-mediated interactions with COP1 and SPA1 (Lian et al., 2011; Liu et al., 2011; Wang et al.,
EP
2001; Yang et al., 2001). CCT2 also mediates CRY2-COP1 interaction (Wang et al.,
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2001). The functional region of CCT2 is further confined to NC80 motif (residues 486-565), which is proposed to function through similar mechanisms to CCT2 (Yu et al., 2007). Interestingly, CCT2 doesn’t mediate CRY2-SPA1 interaction, which is mediated by CNT2 (Zuo et al., 2011). CRY2 is also found to interact with a family of bHLH transcription factors, cryptochrome-interacting bHLH (CIBs) (Liu et al., 2008), through photolyase homology region (PHR, residues 1-498) (Kennedy et al., 2010), which comprises CNT2 (Sang et al., 2005; Yang et al., 2000) and a part of NC80 (Yu 2
ACCEPTED MANUSCRIPT et al., 2007). However, it remains to be determined whether CNT2 or a part of NC80 mediates CRY2-CIBs interactions. In addition, no direct evidence shows whether CRY2 is able to promote flowering independent of CCT2/NC80-mediated signaling.
RI PT
CRY1 and CRY2 sequence alignment reveals a region within CCT1 (residues 489-619) showing similarity to CRY2-NC80, which is here named CRY1-NC80 and doesn’t overlap with CNT1 (Supplemental Figures 1A and 1B). Given that CCT1 and
SC
likely its NC80 are involved in CRY1 signaling, whether CNT1 alone is able to
M AN U
mediate blue-light inhibition of hypocotyl elongation remains unknown. To explore the potential function of CNT1, we sought to make transgenic cry1 mutant lines overexpressing CNT1 and examine their hypocotyl elongation phenotype in blue light. In our previous study, overexpression of CNT1 without the nuclear
TE D
localization signal sequence (NLS) tag in wild type background leads to a dominant negative phenotype under blue light, but fails to confer a phenotype in cry1 mutant background (Sang et al., 2005). Since CRY1 is shown to be localized in both
EP
cytoplasm and nucleus and the nuclear localization is required for CRY1 regulation of
AC C
hypocotyl elongation (Wu and Spalding, 2007), the failure for CNT1 to mediate a light response in cry1 mutant background might result from insufficient nuclear abundance of CNT1. Therefore, we generated transgenic cry1 mutant plants overexpressing CNT1-NLS-YFP (35S::CNT1-NLS-YFP or CNT1; Figure 1A) and the control GUS-NLS-YFP (35S::GUS-NLS-YFP or GUS; Supplemental Figure 2A) fusion protein, respectively. All the GUS lines examined that had strong nuclear YFP signals showed a cry1-like phenotype. Of 80 independent CNT1 lines in T1 examined, 3
ACCEPTED MANUSCRIPT 15 lines without YFP signals exhibited no phenotype in blue light, whereas the remaining 65 lines with nuclear YFP signals displayed varied degrees of shortened hypocotyls, of which 34 lines with the strongest YFP signals hardly elongated, and
RI PT
exhibited extremely compact morphology without elongated stem after transferred to soil and eventually died (Supplemental Figure 2B). We failed to obtain homozygotes of even weak CNT1 lines likely due to impaired fertility. Therefore, we selected three
SC
weak heterozygous lines, CNT1#8, #9, and #13, for detailed phenotypic analyses.
M AN U
All these three lines segregated three classes of siblings in T2, which showed strong, intermediate, and no hypocotyl phenotype, respectively, at a ratio of about 1:2:1, and the results for CNT1#9 were shown (Figures 1B and 1C). The expression of CNT1 was strictly correlated with short-hypocotyl phenotype in all these three lines
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(Figure 1D). Moreover, the siblings segregated from CNT1#9 that had strongly shortened hypocotyls expressed more CNT1 protein than those that had intermediately shortened hypocotyls, while those that had cry1-like hypocotyls didn’t
EP
express CNT1 (Figure 1E), indicating that CNT1 inhibits hypocotyl elongation in a
AC C
dose-dependent manner in blue light. Intriguingly, CNT1 failed to confer a phenotype in either darkness or red or far-red light (Figures 1F and 1G and Supplemental Figures 3A-D), indicating that CNT1 inhibits hypocotyl elongation in a blue-light-dependent manner. Besides the hypocotyl phenotype, we also observed multiple developmental defects for CNT1 plants, including reduced plant size and height, decreased silique length, sterility, and increased shoot branching (Supplemental Figures 2B-E). Notably, of the twenty-two missense mutations in hy4/cry1 alleles, thirteen 4
ACCEPTED MANUSCRIPT mutations occur within the N terminus (Ahmad et al., 1995; Shalitin et al., 2003), demonstrating the functional importance of this domain. We generated transgenic cry1 plants overexpressing mutant CNT1-NLS-YFP corresponding to eight missense cry1
RI PT
alleles, respectively (Figure 1A). In contrast to CNT1 seedlings, all the T1 transgenic seedlings harboring any one of the eight mutant CNT1 transgenes displayed cry1-like long hypocotyls in blue light. Low levels of mutant CNT1 proteins were uniformly
SC
detected in multiple lines for each one of mutant CNT1 transgenes except for
M AN U
CNT1-A462V (corresponding to cry1-305) and CNT1-G283E (corresponding to hy4-5) transgenes. All the representative lines for mutant transgenes exhibited a similar phenotype to cry1 mutant in blue light, of which only CNT1-A462V#4 and CNT1-G283E#3 lines expressed similar levels of CNT1 proteins to CNT1#9 (Figures
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1H-J). We obtained similar results when transiently expressing mutant CNT1s in tobacco leaves (Supplemental Figure 4). Subcellular localization analyses showed that, regardless of the N- or C-terminal YFP fusion, CRY1-A462V and CRY1-G283E
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displayed similar cytoplasmic and nuclear localization patterns to CRY1 in
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Arabidopsis protoplasts or epidermal cells of tobacco leaves (Supplemental Figures 5 and 6), indicating that A462V and G283E mutations likely interfere with CRY1 activity rather than subcellular localization. Taken together, these data indicate that the mutations within CNT1 may affect protein stability and/or properties, thus eliminating its capacity to confer enhanced blue light inhibition of hypocotyl elongation in a manner strikingly similar to the effects of these mutations on the full-length CRY1. To further confirm CNT1 function, we placed YFP at the N terminus of CNT1 5
ACCEPTED MANUSCRIPT and CNT1 mutants (CNT1-A462V and -G283E) and transformed them into cry1 mutant, respectively (Supplemental Figure 7A). We observed that overexpression of YFP-NLS-CNT1 resulted in varied degrees of short-hypocotyl phenotypes in blue
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light, whereas that of CNT1-A462V and CNT1-G283E failed to inhibit hypocotyl elongation (Supplemental Figures 7B-E). These results together with those from CNT1-NLS-YFP overexpression indicate that CNT1 overexpression inhibits
then
performed
RNA-seq
analyses
with
dark-grown
WT,
and
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We
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hypocotyl elongation regardless of N- or C-terminal YFP fusion.
blue-light-grown WT, cry1 cry2, CNT1#9, and CNT1-G283E#3 seedlings. The results showed that CRY regulated a large number of blue light-controlled genes in a strikingly similar manner (3,436, designated CRY-regulated genes; Supplemental
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Figure 8 and Supplemental Table 1). Notably, CNT1 influenced about one third (1,151) of CRY-regulated genes (Figure 1K and Supplemental Table 1), and regulated 998 (87%) genes in the same direction as CRY did (Figure 1L). Gene Ontology (GO)
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analyses demonstrated that CNT1 and CRY are highly involved in the regulation of
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multiple overlapping biological processes including cell wall remodeling and growth-related phytohormones signaling (Supplemental Figure 9). These results indicate that CNT1 is involved in mediating CRY1 signaling. We then compared CNT1-regulated genes with the previously-identified genes
responsive to auxin, brassinosteroids (BR), and gibberellins (GA), respectively. The results showed that the majority of overlapping genes were regulated in an opposite direction by CNT1 and each one of these three phytohormones, auxin (83/116, 72%), 6
ACCEPTED MANUSCRIPT BR (356/488, 73%), and GA (86/108, 80%), respectively (Figures 1M-O and Supplemental Tables 2-4), indicating that the antagonistic regulation of hypocotyl elongation by CNT1 and these phytohormones is likely mediated through antagonistic
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regulation of gene expression. Next, we performed qRT-PCR to confirm CRY and CNT1 regulation of genes that are responsive to auxin, BR, and GA by selecting ten representative genes, whose
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expression is shown to be up-regulated by at least one of auxin, BR, and GA.
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Moreover, the selected SAUR19-SAUR24 subfamily genes, SAUR63, PRE1, PRE5, EXP8, and HAT2, are shown to promote hypocotyl elongation. We found that the expression of each one of these ten genes was drastically more reduced in WT seedlings grown in blue light than in those grown in darkness, while less inhibited in
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cry1 cry2 or cry1 than in WT in blue light (Figure 1P and Supplemental Figure 10). Strikingly, CNT1 overexpression considerably reduced the expression of these genes, whereas CNT1-G283E or CNT1-A462V overexpression did not (Figure 1P and
at
least
in
part,
through
CNT1-mediated
repression
of
the
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light,
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Supplemental Figure 11), indicating that CRY1 inhibits hypocotyl elongation in blue
auxin/BR/GA-responsive gene expression. CNT1 may confer blue light response by working together with CRY2 to inhibit
COP1-SPA complex through CCT2 and/or by interacting with unidentified factors to mediate pathways that are totally separate from COP1/SPA-mediated pathway. Since CNT1 overexpression in wild type background leads to dominant negative effects on endogenous CRY1 through CNT1-CRY1 interaction (Sang et al., 2005), the enhanced 7
ACCEPTED MANUSCRIPT blue light response conferred by CNT1 in cry1 may not result from the enhanced CRY2 activity by CNT1-CRY2 interaction. Indeed, CNT1 overexpression in cry1 cry2 double mutant resulted in varied degrees of shortened hypocotyls in blue light
enhanced
accumulation
of HY5,
whereas
CNT1
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(Supplemental Figures 12A and 12B). Interestingly, CCT1 overexpression led to overexpression
did
not
(Supplemental Figure 13). These results strongly indicate that CNT1-mediated inhibition
of hypocotyl
elongation
does
not
proceeds
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blue-light
through
(Supplemental Figure 14).
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CCT1/CCT2-COP1/SPA-HY5 pathway, but likely through totally new pathways
We revealed a previously unrecognized function of CNT1 in mediating light responses in Arabidopsis, which does not require CCT1, thus advancing our
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understanding of CRY1 signaling mechanism and opening up a new avenue for exploring CRY1-interacting proteins. Great efforts should be made to characterize the potential CNT1-interacting proteins in future study. Given that Arabidopsis CRY2
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interacts with transcription regulators likely through CNT2 (Kennedy et al., 2010) and
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that type I animal CRY is clearly shown to mediate signaling through its N terminus-mediated interactions with transcription regulators (Busza et al., 2004), as well as that CNT1 is implicated in the inhibition of auxin/BR/GA-responsive gene expression (Figures 1M-P), it will be worth exploring whether CNT1 might directly regulate the transcription regulators in auxin/BR/GA signaling pathways.
FUNDING 8
ACCEPTED MANUSCRIPT This work was supported by grants from the National Natural Science Foundation of China (90917014, 91217307, and 30830012 to H.-Q.Y., and 31170266 to H.-L.L.).
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China Innovative Research Team, Ministry of Education, and 111 Project (B14016).
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Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin
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FIGURE LEGENDS
Figure 1. CNT1 alone is sufficient to inhibit hypocotyl elongation. (A) Schematic diagram depicting the construct expressing CNT1-NLS-YFP used for transformation of cry1 mutant. Various point mutations are also indicated. NLS indicates nuclear localization signal. 11
ACCEPTED MANUSCRIPT (B, F, and H) Phenotypes of 5-day-old seedlings. All the lines harboring the constructs shown in (A) are in cry1 background. S, M, and L denote short, intermediate, and long hypocotyls of siblings segregated from heterozygous CNT1#9
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line (B), respectively. Dk (F) and BL (B and H) denote darkness and blue light (30 µmol/m2/s for B; 20 µmol/m2/s for H), respectively. Bar=2 mm.
(C and I) Statistical analyses of hypocotyl length corresponding to (B and H),
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respectively. The numbers of segregated seedlings for each phenotype were shown
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above each column (C). Data are means ± SD (n was shown above each column for C; n=30 for I).
(D, E, G, and J) Western blot analysis showing CNT1 protein accumulation. S and L denote samples from blue-light-grown siblings with short and long hypocotyls
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segregated from the indicated heterozygous CNT1 lines, respectively (D). The results shown in (E), (G), and (J) correspond to samples from seedlings shown in (B), (F), and (H), respectively.
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analyses.
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(K) Venn diagram showing overlapping genes among datasets from RNA-seq
(L) Hierarchical clustering analysis of 1,151 overlapping genes as shown in (K). (M-O) Venn diagrams showing the overlapping genes between datasets and hierarchical clustering analyses showing largely converse regulation of overlapping genes by CNT1 and auxin (M) or brassinosteroids (BR, N) or gibberellins (GA, O). The scale bar indicates the log2 value of fold change (L-O). (P) qRT-PCR analysis showing the effects of CNT1 and CNT1-G283E on 12
ACCEPTED MANUSCRIPT auxin/BR/GA-responsive gene expression. Blue-light-grown seedlings with the indicated genotypes were used. The relative expression was normalized to that in WT arbitrarily set to 1. Data are means ± SD (n=2 for IAA5 and SAUR15; n=3 for other
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genes).
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