Chapter 6: ZBFl acts as a negative regulator of photomorphogenesis and light regulated gene expression in Arabidopsis

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1 Chapter 6: ZBFl acts as a negative regulator of photomorphogenesis and light regulated gene expression in Arabidopsis

2 6.1 Introduction The growth and development of plant is controlled by several environmental factors, of which light is arguably the most important one (Kendrik and Kronenberg, 1994; Deng and Quail, 1999; Neff et ai., 2000; Quail 2002). The shift from skotomorphogenic to photomorphogenic development leads to a change in expression of about one-third of the total genes in Arabidopsis (Tepperman et ai., 2000; Ma et ai., 2001). Several transcription factors in light signaling pathways have been reported that are involved in photomorphogenic development. HY 5 is the first genetically defined bzip transcription factor in light signaling pathways (Koornneef et ai., 1980; Oyama et ai., 1997; Chattopadhyay et ai., 1998a). The hy5 mutant seedlings show partially etiolated phenotype in red, far red, or blue light, and have more lateral roots as compared to wild type plants (Koornneef et ai., 1980; Ang and Deng, 1994; Oyama et ai., 1997; Pepper and Chory, 1997). It has been demonstrated recently that auxin plays an important role in HY5 mediated root development (Cluis et ai., 2004). Recently, a similar bzip protein, HYH has been reported, mutation in which leads to blue light specific partial etiolation (Holm et ai., 2002). Mutations in bhlh protein HFRlIREPlIRSF1 lead to an etiolated phenotype only in the far red light (Fairchild et ai., 2000; Soh et ai., 2000; Spiegelman et ai., 2000). Two other bhlh proteins, PIF3 and PIF4, have been shown to be involved in phytochrome mediated transcriptional regulation. Furthermore, it has been demonstrated that phyb interacts with the G-box bound PIF3 (Ni et ai., 1998). Mutational studies have recently shown that PIF3 negatively regulates phyb mediated inhibition in hypocotyl elongation (Kim et ai., 2003). LAF1, a MYB protein, has been shown to be involved in far red light mediated signaling (Ballesteros et ai., 2001). Two other MYB proteins, LHY and CCA, are involved in circadian rhythm (Schaffer et ai., 1998; Wang and Tobin 1998). The crosstalk of signaling pathways in plant has just started to be unraveled. Cell cyele genes, for example cyelin dependent kinases, are regulated by salt stress (Burssens et ai., 2000; Hirt, 2000). The Arabidopsis DEAD-box RNA helicase mutant los4 is chilling sensitive and impaired in the cold-regulated expression of CBF genes (Gong et ai., 2002). Phytochrome mediated light signaling has recently been demonstrated to be involved in the regulation of TOP2, one of the components of DNA replication and cell 52

3 cycle machinery (Hettiarachchi et ai., 2003). Interestingly, a promoter determinant, ClDRE, which is known to respond to low temperature, has been shown to be involved in phyb mediated light signaling to cold-induced gene expression (Kim et ai., 2002). Using studies with Arabidopsis mutants affected in light perception, it was recently shown that phytochrome signaling interacts with salicylic acid (SA) signal transduction (Genoud et ai., 2002). Weatherwax (1996, 1998) earlier demonstrated an interaction of light and ABA in the regulation of plant gene expression in Lemna gibba. ABA plays an important role in the regulation of plant water balance and osmotic stress tolerance (Yamaguchi Shinozaki and Shinozaki, 1994; Leung and Giraudat, 1998; Finkelstein and Lynch, 2000). AtMYC2 is a bhlh transcription factor, which has been shown to be functioning as an activator in ABA signaling pathways (Abe et ai., 2003). Very recently, it has also been demonstrated that AtMYC2 (JIN1) acts in jasmonic acid (JA) signaling pathways in Arabidopsis and tomato (Boter et ai., 2004; Lorenzo et ai., 2004). In this chapter, we further demonstrate that AtMYC2 (ZBF1) is involved in light regulated gene expression and photomorphogenic development in Arabidopsis. It was previously shown by DNAprotein interaction studies that Z-box binding factor (ZBF) activity was present in Arabidopsis (Yadav et ai., 2002). Here, we demonstrate the function of ZBFl (AtMYC2/JIN1) in light signaling pathways. Our genetic analyses suggest that ZBFl acts as a negative regulator of blue light mediated photomorphogenic growth and gene expression. Taken together these results demonstrate that ZBFl is a transcription factor, which functions in ABA, JA and light signaling pathways in Arabidopsis thaliana. 53

4 6.2 Results Isolation and characterization of a null mutation in ZBFl Since ZBFl interacts with the Z-box LREs present in the light regulated promoters of CABl and RBCS-lA, respectively, we ask whether ZBFl is involved in the regulation of photomorphogenic growth in Arabidopsis. To address this question, we searched for mutants in T -DNA knockout collections (Alonso et ai., 2003). A mutant line with T -DNA insertion at the 5' end of ZBF 1 coding sequence was identified and the corresponding allele was designated as zbfl-l. The junctions of T -DNA and ZBF 1 were amplified by PCR and the DNA sequence analyses revealed that the T -DNA was inserted in nucleotide position 960bp from the start codon (Figure 1). Northern blot, RT-PCR and Western blot analyses were unable to detect any transcript or protein encoded by ZBF 1 in zbfl-l mutant background (Figure 2). Therefore, the T -DNA insertion in ZBF 1 likely caused instability of the corresponding transcript and resulting in a null mutation. Since the other T -DNA tagged mutant lines identified were not null for the ZBF 1 mutation, we chose to characterize the zbfl-l mutant line zbfj-l exhibits blue light specific morphological defects in seedling development We measured the hypocotyl length of 6-day-old zbfl-l and wild type seedlings grown under constant dark or WL conditions. However, no difference in hypocotyl elongation was detected between wild type and zbfl-l mutants grown in constant darkness or WL conditions (Figure 3). To determine whether the zbfl-l mutants have any altered morphology in a particular wavelength of light, we examined the growth of 6-day-old seedlings under various wavelengths of light such as RL, FR and BL. Whereas the enhanced inhibition in hypocotyl elongation of zbfl-l was observed in constant BL, no significant change in hypocotyllength was observed in constant FR or RL (Figure 4A-C). Measurements of hypocotyl length revealed that 6 day old BL grown zbfl-l mutant seedlings had significantly shorter hypocotyls as compared to wild type seedlings (Figure 7 A). Although FR grown zbfl-l mutants did not show any altered morphology, the 54

5 A 882bp ~ 1 RP2 LP2 1900bp IATG ~ 590bp Z LBP~ 292bp I B 882 bp 590 bp Figure 1.T-DNA insertion site in ZBF1 and Genomic per analysis. A, T-DNA insertion at 960bp from the start codon. The size of the PCR products with LP2, RP2 and LBP are shown by arrows. B, PCR reactions were carried out using gene specific primers LP2 and RP2 and T-DNA primer LBP. Lane 1 and 4 show the PCR results with wild type chromosomal DNA as the template. Lanes 2,3, 5 and 6 show the PCR results with the chromosomal DNA of zbf1-1 homozygous mutant as template.primers used in lane 1,2 and 3 were LP2, RP2 and LBP. Primers used in lane 4, 5 and 6 were LP2 and RP2. Lane number 7 shows the molecular weight marker (21, 5, 3, 2, 1.8, 1.5, 1.3,0.95, 0.83, 0.56 kb ).

6 ~ 960 bl2 ~ ~ B C D ZBF ZBF1 ZBF1 1 kb rr 500bp Actin *" Col zbf1-1 Col zbf Figure 2. Identification of a T-DNA tagged mutation in ZBFl gene. A, The schematic diagram of the T-DNA insertion site in ZBF 1. The inverted triangle shows the T-DNA insertion site after 960 bp from the start codon. B, Northern blot of 20 ~g of total RNA isolated from 6 days old WL grown wild type (col) and zbj1-1 mutant seedlings. C, RT-PCR analysis of wild type and zbj1-1 mutant using gene specific primers LP2 and RP2. Actin primers were used as control. D, Western blot of 10 ~g of total protein extracted from 6 days old WL grown wild type and zbj1-1 mutant seedlings. The asterisk marks a cross-reacting protein band indicating the loading control.

7 Figure 3. Six-day-old constant dark (0) or white light (WL) grown seedlings. In each panel, wild type and zbf1-1 mutants seedlings are shown on theleft and right side, respectively.

8 mutant seedlings had high level accumulation of anthocyanin at the junction of hypocotyls and cotyledons (Figure 4C), a characteristic of hyperphotomorphogenic growth (Ang et ai., 1998). Taken together these results suggest that ZBFl acts as a negative regulator of photomorphogenesis and its effect is more pronounced under BL condition Mutations in ZBFl results in pleitropic effects We asked whether the zbfl-l mutants have any altered morphology in the adult stage. Examination of root growth of zbfl-l mutant plants revealed that 16-day-old mutant plants developed significantly less lateral roots as compared to wild type plants (Figure 5). Furthermore, whereas zbfl-l mutant seedlings did not exhibit any altered morphology while grown in various fluences of WL, the mutant adult plants exhibited significantly short stature as compared to WL grown wild type plants (Figure 6). Taken together these results suggest that mutations in ZBFl results in multiple effects Characterization of zbfj-l mutants Light signaling controls various physiological processes through the regulation of light responsive genes (Ma et ai., 2001; Tepperman et ai., 2001; Wang et ai., 2002). The accumulation of chlorophyll and anthocyanin are two such important physiological responses. To determine whether ZBF 1 has any role in chlorophyll accumulation, we quantified the chlorophyll content in wild type and zbfl-l mutant seedlings. As shown in Figure 7B and C, the chlorophyll and anthocyanin contents were significantly higher in zbfl-l mutants as compared to wild type seedlings. While propagating zbfl-l mutant plants, we observed that zbfl-l mutation caused late flowering. Whereas long-day-grown wild type plants start flowering after the formation of about 8 rosettes, the zbfl-l mutants flower after 11 rosettes formed (Figure 7D) ~BFl negatively regulates the expression of light inducible genes To determine the role of ZBFl in the regulation of light inducible gene expression, we performed RNA gel blot analyses and measured the expression of CAB, 55

9 Figure 4. Six days old various light grown zbf1-1 mutant seedlings. In each panel wild type and zbf1-1 mutant seedlings are shown on the left and right, respectively. A, RL grown seelings. B, BL grown seedlings. C, FR grown seedlings.

10 Figure 5. Root phenotype of zbf1-1. Wild type and zbf1-1 mutant are shown on left and right, respectively. Twelve days old seedlings grown in constant WL are shown.

11 A B WT zbjl-l WT zbjl-l Figure 6. Phenotype of wild type and zbf1-1 mutant plant at early and late adult stage. In each panel wild type and the zbfl-l mutant plants are shown on the left and right panels, respectively. A, Twenty one days old plants of wild type and zbfl-l mutant plant are shown. B, Thirty five days old wild type and zbfl-l mutant plants.

12 A 6 - E 5.s 4 O'l c ~ 3 >. (5 2 u o g;1 I o -'---'---'--'---'-- Col zbf1-1 B~500 Q) :J rn ~ 400.g> Cl g 300 CD ~ 200 >,..c 0.. e 100 o :c () 0.L...L_J...L..----'-_ Col zbf1-1 C 0.5 Q) ~ 0.4 rn ~.g> r:: 0.3 J ~ 0.2 c:i '~ 0.1 :: "'--'-_~_.L- Col zbf1-1 Cl ~ o.c iii 10 rn ~ «I Q) = o 5 c:i Z o -'--'-"'-' Col zbf1-1 Figure 7. Characterization of zbf1-1 mutants. A, six days old constant BL grown (30!-lmol/secl m 2 ) seedlings were used to measure the hypocotyl length.the error bars indicate the standard deviation. B, Accumulation of chlorophyll a and b in wild type and zbf1-1 mutant seedlings grown under constant BL (30!-lmol/secl m 2 ). C, Accumulation of anthocyanin in wild type and zbf1-1 mutant seedlings grown under constant BL(30!-lmol/secl m 2 ). D, Number of rosette leaves at bolting in long day cycle of 16h white light (100!-lmol/secl m 2 ) and 8h darkness.

13 RBCS and CHS genes in 6-day-old various light grown seedlings. As shown in Figure SA, the expression of the light inducible genes was significantly elevated in zbjl-l mutants as compared to wild type seedlings in BL and FR. In the case of RBCS, about two fold increase in the transcript level was detected in BL, however, the expression of the gene was found to be 3 fold increased in zbjl-l mutant background in the FR (Figure SB). Whereas very little increase, if any, in the expression of CHS and CAB was detected in zbjl-l mutants in WL, about 2-3 fold increase was detected in BL and FR grown mutant seedlings as compared to wild type background (Figure SC and D). No significant change in expression in RL was detected in between wild type and zbjl-l mutant backgrounds (data not shown). Taken together these results suggest that ZBFl acts as a negative regulator of light regulated gene expression in BL and FR specific manner zbfj-l mutants are less sensitive to ABA and JA responsiveness It was previously shown that mutation in AtMYC2 (generated by AciDs tagging system) caused Arabidopsis plants to be less sensitive to ABA (Abe et ai., 2003). Furthermore, it has been recently demonstrated that jinl-l mutants are less sensitive to JA (Lorenzo et ai., 2004). To determine whether zbjl-l mutants respond to ABA and JA in a similar fashion, we monitored the effect of ABA and JA on zbjl-l mutant plants. Seeds of wild type and mutant plants were plated on MS plates with or without various concentrations of ABA. As shown in Figure 9B, whereas I!lM ABA severely reduced the rate of germination of wild type seeds, the effect was significantly suppressed in zbji-l mutants. However, no noticeable effect of ABA on growth of the zbjl-l mutants was observed as compared to wild type plants (Figure 9C). It has been reported very recently that mutations in lin 1 results in insensitivity to JA mediated root growth retardation (Lorenzo et ai., 2004). To determine the effect of JA on the root growth of zbji-l mutant plants, we grew wild type and zbjl-l mutant plants in the presence of 20!lM JA and monitored the root growth. JA caused severe root growth retardation in wild type plants, however the effect was drastically reduced in zbjl-l mutant plants (Figure 10). These results altogether indicate that zbjl-l mutants are less sensitive to ABA and JA mediated signaling. To determine whether the ABA and JA mediated effects are light specific, we carried out the above experiments in various light 56

14 A B ~ so RBGS > <D 0 WT U1I zbf1-1 GHS u en ffi 40 '- P"""" GAB 18S Cen SO ~. A2' 1 ' WT M WT M WT M WT M o WL BL FR c -<D > :;=; 20 cts <D a: Orml Denso <D <D > 0 WT > <D <D 0 WT 0.60 lid zbf lid zbf1-1 c c u u en en ffi 40 ffi 40 -'- '- r- <D <D.;::; > 20.;::; > 20 co cts - <D <D a: 0 - I rttti, a: WL BL FR 0 WL BL FR GHS GAB - I 0 WL BL FR RBGS I Figure 8. Light regulated gene expression. A, Six-day-old Dark (D), white light (WL), blue light (BL) or far red light (FR) grown seedlings were used for Northern blot analyses. B, C and 0, Quantification of data in A, by Flour-S-Multi Image (Biorad).

15 Figure 9. The effect of ABA on zbf1-1 mutant.ln each panel WT and the mutants are shown on left and right, respectively. A, six days old constant WL grown seedlings without ABA. B, six days old constant WL grown seedlings with 111M ABA. C, Twelve days old constant WL grown seedlings with 111M ABA. (WT: wild type)

16 Figure10. Effect of JA on zbf1-1mutants. Wild type and the zbf1-1 mutant are shown on left and right, respectively. Fifteen days old constant WL grown seedlings with 20J.lM JA.

17 conditions including the blue light where the effect of mutations in ZBF 1 is prominent. However, our results indicate that the less sensitivity of zbfl-l mutants to ABA and JA signaling is not BL specific. 57

18 6.3 Discussion Whereas several photomorphogenesis promoting regulators have been reported in light signaling pathways, very few have been reported to be acting as repressor in a light specific manner. Here, we have reported a blue light specific repressor of photomorphogenic growth. Mutational studies with zbfl-l highlight the existence of cross-talk among light, ABA and JA signaling, and thus establishes a functional relationship among these signaling pathways. Three downstream signaling components in blue light, HYH, AtPP7 and SUBl, have been reported earlier. Whereas HYH and AtPP7 act as positive regulators of blue light mediated photomorphogenic growth, SUBI acts as a negative regulator of blue light and far red light mediated signaling (Guo et ai., 2001; Holm et ai., 2002; Moller et ai., 2003). The analyses of zbfl-l mutants clearly demonstrate that the short hypocotyl phenotype of zbfl-l seedlings is restricted to BL. These results suggest that although ZBF 1 is expressed in the dark and various light grown seedlings, it functions as a negative regulator of BL specific photomorphogenic growth mediated by cryptochromes. Analyses of the light regulated gene expression in zbfl-l mutants further reveal that ZBFl represses the blue light mediated expression of CAB, RBCS and CHS genes. Furthermore, although zbfl-l mutants do not exhibit any morphological defects in far red light, the light regulated genes are upregulated in far red light in zbfl-l mutant background. These results demonstrate that ZBFl plays a negative regulatory role in the expression of light inducible genes in blue light and far red light specific manner. It has already been demonstrated that ZBFl (AtMYC2/JINl) acts as a transcriptional regulator in ABA and JA signaling pathways (Abe et ai., 2003; Lorenzo et ai., 2004). We have examined the ABA and JA responsiveness of zbfl-l mutants and our results demonstrate that the zbfl-l mutant plants are partially insensitive to ABA and la. However, the compromised sensitivity of zbfl-l mutants to ABA and JA is not specific to a particular wavelength of light. Abe et ai., 2003 have reported that mutations in AtMYC2 results in better growth in the presence of ABA as compared to wild type plants. However, our studies with zbfl-l mutants were unable to detect the corresponding effect. It is possible that this effect is more prominent at the adult stage (as found by Abe et ai., 2003) rather than the 12-day-old plants. Studies with copl mutants have revealed that 58

19 COPl, a master repressor of photomorphogenic growth in the darkness, acts as a positive regulator of lateral root formation (Ang et ai., 1998). Analyses of zbfl-l mutants in this study have revealed that although ZBFl is a negative regulator of blue light mediated photomorphogenic growth, it is essential for lateral root formation. Several light signaling components have been described previously, which function as positive as well as negative regulator of light responses (Deng et ai., 1991; Wang et ai., 1997; Chattopadhyay et ai., 1998b; Liu et ai., 2001). For example, PIF3, a phytochrome interacting bhlh protein, acts as a positive regulator for CHS induction however, negatively regulates the inhibition of hypocotyl elongation, cotyledon opening and expansion (Kim et ai., 2003). We have demonstrated that ZBFl is a negative regulator of BL mediated photomorphogenic growth, and blue and far red light regulated gene expression (Figure 11), however it acts as a positive regulator of lateral root formation. Furthermore, whereas ZBFlIAtMYC2/JINI acts as a positive regulator of ABA signaling, it plays both positive and negative regulatory roles in JA signaling pathways. The exact mechanism of ZBFlIAtMYC2/JINI mediated differential regulation of signaling is not known. A simple way to explain the differential regulation is to consider that ZBFl could function either as a transcriptional activator or repressor,.-~ -. depending on the specific promoter determinants of target genes. Alternatively, extensive hetero-dimerization of bhlh proteins have been reported. Therefore, it could be envisioned that hetero-dimerization of ZBFl with other bhlh proteins might be a potential mechanism to generate positive and negative regulators, which in turn play opposite roles in signaling cascades. Whereas JA signaling pathways are poorly understood, ABA signaling pathways have been investigated to some detail. Potentially, light and ABA effects are antagonistic: for example, (i) suppression of seed germination by ABA is enhanced in the light (Fellner and Sawhney, 2002); (ii) light grown seedlings accumulate ABA when transferred to darkness and brief red-light pulses decrease ABA amounts (Weatherwax et ai., 1996); and (iii) ABA mutants show altered responses to photoperiod and light quality (Rohde et ai., 2000; Fellner et ai., 2001; Fellner and Sawhney, 2002). Therefore, plants have evolved the ability to integrate various signals and to respond accordingly in a comprehensive manner. Demonstration of ZBFl as a common transcriptional regulator 59

20 Blue Light \,.... E, Far red Light..... Photomorphogenic growth ---II LlGs ZIG-box L-_'-;"'~_"" Figure 11. Model showing the functional involvement of ZBF1 in blue light mediated signaling. ZBF1 acts as a repressor of blue light mediated photomophogenic growth. It also acts as a negative regulator for blue and far red light regulated gene expression. LlGs indicates light inducible genes.

21 for light, ABA and JA signaling establishes a functional relationship among them and thus will help to decipher the mechanism of integration of these signaling pathways in future studies. 60