Functional interconnections of HY1 with MYC2 and HY5 in Arabidopsis seedling development

Abstract

Arabidopsis seedling development is controlled by many regulatory genes involved in multiple signaling pathways. The functional relationships of these genes working in multiple signaling cascades have started to be unraveled. Arabidopsis HY1/HO1 is a rate-limiting enzyme involved in biosynthesis of phytochrome chromophore. HY5 (a bZIP protein) promotes photomorphogenesis, however ZBF1/MYC2 (a bHLH protein) works as a negative regulator of photomorphogenic growth and light regulated gene expression. Further, MYC2 and HY1 have been shown to play important roles in jasmonic acid (JA) signaling pathways. Here, we show the genetic interactions of HY1 with two key transcription factor genes of light signaling, HY5 and MYC2, in Arabidopsis seedling development. Our studies reveal that although HY1 acts in an additive manner with HY5, it is epistatic to MYC2 in light-mediated seedling growth and gene expression. This study further demonstrates that HY1 additively or synergistically functions with HY5, however it works upstream to MYC2 in JA signaling pathways. Taken together, this study demonstrates the functional interrelations of HY1, MYC2 and HY5 in light and JA signaling pathways.

Figure 1

The ehy5 mutants display elongated hypocotyl. A, Phenotype of segregated wild-type (WS), ehy5, hy5, and ehy5 hy5 double mutants in dark and different light conditions. Six-day old constant dark, WL (90 μmol m^-2 s^-1), FR (90 μmol m^-2 s^-1), RL (90 μmol m^-2 s^-1) and BL (45 μmol m^-2 s^-1) grown seedlings (a to e). B-C, Quantification of hypocotyl length of 6-day-old seedlings grown in constant dark and various fluences of WL, respectively. D-F, Quantification of hypocotyl length of 6-day-old constant FR (90 μmol m^-2 s^-1), RL (90 μmol m^-2 s^-1) and BL (45 μmol m^-2 s^-1) grown seedlings, respectively. The error bar indicates standard deviation (SD). The experiment was repeated more than twice and similar results were obtained each time. A representative result is presented. For measuring hypocotyl length, ~30 seedlings were used in each genotype.

Figure 2

Positional cloning and molecular identification of EHY5. A, Map-based cloning. The genetic locus of ehy5 mutation was first mapped between markers ER and T20P8 on chromosome 2. Fine mapping using genetic markers designed from BAC clones. The direction of the BAC clone is indicated by the arrow. The numbers above and below the arrow indicate the marker number and the corresponding recombinants for the respective marker. Sequence of the genomic DNA fragment from wild-type (WS) and ehy5 mutant plants and comparison with wild-type (Col) genomic DNA sequence indicate C to T mutation. B, DNA polymorphism between ehy5 and wild-type (WS) plants. The C to T mutation in ehy5 genomic DNA adds a DdeI recognition site. The DNA fragments flanking the DdeI site were amplified from the wild-type and ehy5 plants, digested with DdeI, and separated on native PAGE. C, Genetic complementation of ehy5. Phenotypes of 6-day-old wild-type (WS), ehy5 and ehy5* (HY1 complemented) are shown. D, RT- PCR results show the expression of HY1 in wild-type (WS), ehy5 and ehy5*. Actin bands show the loading control. The RT-PCR experiment was repeated thrice and a representative result is shown.

Figure 3

HY1 and HY5 additively regulate the light-induced gene expression. A - B, Relative expression of CAB1and RBCS-1A in 6-day-old seedlings grown in WL (90 μmol m^-2 s^-1). C, Accumulation of chlorophyll in 6-day-old wild-type and mutant seedlings grown in WL (90 μmol m^-2 s^-1). D, Accumulation of anthocyanin in 6-day-old wild-type and mutant seedlings grown in WL (90 μmol m^-2 s^-1). The error bars indicate SD. *** - indicates significant difference from hy5 (p > 0.001 student's t-test, n = 30, number of seedlings used for hypocotyl measurement). Real-time PCR was repeated more than thrice and in each biological experiment three technical replicates were used. Similar results were obtained in all the experiments. A representative figure is shown here. For chlorophyll and anthocyanin estimation, 50 seedlings was used in each genotype and the experiment was repeated thrice and in each biological experiment, four technical replicated were used. Similar results were obtained in all the experiments. A representative figure is presented.

Figure 4

JA responsiveness of hy1 hy5 double mutants. A, Quantification of root length of 16-day-old wild-type and hy1 mutant plants grown in constant WL (90 μmol m^-2 s^-1) without hormone (JA). B, Quantification of root length of A. C, Relative induction of VSP2 expression by JA in wild-type and mutant plants. Six-day-old wild-type and mutant seedlings were treated with MS (Mock) or with JA (50 μM) for 5-hours and total RNA was isolated from 100 mg of tissue and used for quantitative real-time PCR analysis. ACTIN2 was used as internal control. Approximately 25 to 30 seedlings were used for the root growth measurement. The error bars indicate standard error (SE) of three biological replicates.

Figure 5

JA responsiveness of hy1 atmyc2 double mutants. A, Root growth of 16-day-old wild-type and various mutant plants grown in constant WL (90 μmol m^-2 s^-1) in presence of 15 μM JA. B, Quantification of root length of A. C, Relative induction of VSP2 expression by JA in wild-type and mutant plants. For experimental detail, see legend to Figure 4C.

Figure 6

Light-mediated seedling development of hy1 atmyc2. A, Phenotype of wild-type and various mutant seedlings in different light conditions. Six-day-old constant WL (90 μmol m^-2 s^-1), RL (90 μmol m^-2 s^-1), FR (90 μmol m^-2 s^-1) and BL (45 μmol m^-2 s^-1) grown seedlings. B-E, Quantification of hypocotyl length of 6-day-old constant WL (90 μmol m-² s^-1), RL (90 μmol m^-2 s^-1), FR (90 μmol m^-2 s^-1) and BL (45 μmol m^-2 s^-1) grown seedlings, respectively. F-G, The relative expression of CAB1 and RBCS-1A in 6-day-old seedlings grown in WL (90 μmol m^-2 s^-1). The error bars indicate SD. Approximately 25 to 30 seedlings were used for hypocotyl length measurement. For gene expression studies, total RNA was isolated from 100 mg of tissue was used for cDNA preparation. The real-time PCR experiments were repeated more than twice and three technical replicates were used for each genotype. Similar results were obtained in all the experiments. A representative graph is shown. H, Working model shows the role of HY1, HY5 and MYC2 in photomorphogenesis and JA responsiveness. HY1 and HY5 act additively in response to JA and light signaling pathways. MYC2 acts downstream to HY1 in JA responsiveness, and HY1 acts negatively to MYC2-mediated BL specific photomorphogenic growth.