Real-Time Monitoring of Key Gene Products Involved in Rice Photoperiodic Flowering

Abstract

Flowering is an important biological process through which plants determine the timing of reproduction. In rice, florigen mRNA is induced more strongly when the day length is shorter than the critical day length through recognition of 30-min differences in the photoperiod. Grain number, plant height, and heading date 7 (Ghd7), which encodes a CCT-domain protein unique to monocots, has been identified as a key floral repressor in rice, and Heading date 1 (Hd1), a rice ortholog of the Arabidopsis floral activator CONSTANS (CO), is another key floral regulator gene. The Hd1 gene product has been shown to interact with the Ghd7 gene product to form a strong floral repressor complex under long-day conditions. However, the mRNA dynamics of these genes cannot explain the day-length responses of their downstream genes. Thus, a real-time monitoring system of these key gene products is needed to elucidate the molecular mechanisms underlying accurate photoperiod recognition in rice. Here, we developed a monitoring system using luciferase (LUC) fusion protein lines derived from the Ghd7-LUC and Hd1-LUC genes. We successfully obtained a functionally complemented gene-targeted line for Ghd7-LUC. Using this system, we found that the Ghd7-LUC protein begins to accumulate rapidly after dawn and reaches its peak more rapidly under a short-day condition than under a long-day condition. Our system provides a powerful tool for revealing the accurate time-keeping regulation system incorporating these key gene products involved in rice photoperiodic flowering.

Keywords: circadian clock; luciferase (LUC); photoperiodic flowering; rice; short-day plant.

FIGURE 1

Gene-targeting of LUC gene fused to Ghd7 gene. (A) Outline of experimental strategy of gene targeting. HindIII-cut fragments (gray arrows), BamHI-cut fragments (blue arrows), Ghd7 probe (gray bars; 1273 kb), LUC probe (blue bars; 974 bp), and PCR fragments (red arrows). The gene-targeted Ghd7::Ghd7-LUC (GT,HPTb) line was properly selected by PCR screening (B) and by Southern blot hybridization (C). Primer sequences are listed in Supplementary Table 1.

FIGURE 2

K-mer Analysis (k = 50) in WT and Ghd7::Ghd7-LUC(GT,HPTb) line. It was shown that Ghd7::Ghd7-LUC(GT,HPTb) is a valid GT line by k-mer analysis. The expected sequence for Ghd7::Ghd7-LUC(GT,HPTb) line was used as the target reference DNA sequence for k-mer analysis. 6 Gb illumina fastq data of both WT and Ghd7::Ghd7-LUC(GT,HPTb) line were analyzed to count the perfect match in tested fastq data with each k-mer sequence which is produced by scanning the target reference DNA sequence [Ghd7::Ghd7-LUC(GT,HPTb) line]. Genomic sequences outside the construct (Red region) and sequences in the construct array (Black region). The gaps in WT indicates it doesn’t have LUC, IR, and HPT. Magnified 5′ and 3′ regions where the homologous recombination must have occurred are shown with smoothing using the moving average method for intact k-mer values.

FIGURE 3

The effects of Ghd7-LUC protein on flowering time. The gene-targeted Ghd7::Ghd7-LUC(GT,HPTb) line exhibited normal photoperiodic flowering properly compared with the wild type. This indicates that the Ghd7-LUC protein could mimic the Ghd7 protein at least in terms of the photoperiodic control of flowering in rice. There was no significant difference in the flowering time under long-day (14.5 h of light; p = 0.14) and short-day (10 h of light; p = 0.20) conditions (p < 0.05; Student’s t test). Nipponbare is shown as WT, Ghd7::Ghd7-LUC(GT,HPTb) homo lines are shown as Ghd7-LUC.

FIGURE 4

LUC Protein monitoring system. (A) Outline of experiments for LUC protein monitoring. Day1: Plant seeds are sown on a medium (0.2% Gellan gum + MS + 100 μl of 100 mM luciferin solution). The seedlings are germinated in the constant light incubator for 4 days. Day 4: 1 ml of LUC solution (10 mM) is sprayed. Day 5: the seedlings are put into the monitoring system. The light emissions from an entire growing rice seedling are continuously recorded by a photomultiplier tube (PMT) for 6 days. Day 9: 1 ml of LUC solution (10 mM) is sprayed again. Day 11: Leaves are sampled for quantitative RT-PCR. (B) Left, the monitoring unit consisting of a movable photomultiplier tube (PMT) and a LED lamp (upper right); right, the seedlings in test tubes used for this study (Day 11).

FIGURE 5

Diurnal dynamics of the Ghd7-LUC and Hd1-LUC proteins under long-day (16 h) and short-day (10 h) conditions. The diurnal patterns of Ghd7-LUC (Day 8–10) and Hd1-LUC (Day 6–8). Ghd7-LUC protein patterns in six Ghd7::Ghd7-LUC(GT,HPTb) homo plants under long-day (GL1,GL2,GL3) and short-day (GS1,GS2,GS3) conditions. Hd1-LUC protein patterns in six Hd1::Hd1-LUC(RI,HPTb)-1 homo plants under long-day (HL1,HL2,HL3) and short-day (HS1,HS2,HS3) conditions. The diurnal LUC activity dynamics for each plant were recorded for consecutive 72 h. The representative data from a few independent experiments for both day-length conditions were presented. Shades represent dark periods. (A) The diurnal patterns of Ghd7-LUC and Hd1-LUC proteins for 72 h. (B) The mean and standard error of the three samples in panel (A) are displayed. Shades represent dark periods.

FIGURE 6

Ghd7-LUC and Hd1-LUC Proteins and related mRNA pattern. Scatter plot of LUC proteins activity and mRNA transcription level under long-days and short-days conditions. The amount of LUC proteins indicates monitored values of LUC activity at 1 h after light-on on the day 11. The corresponding amount of mRNA of related genes were shown. The leaves were sampled at 1.5 h after light-on. LUC mRNA was not detected in WT. The transcript levels of RFT1 were too low to be detected in some lines (showed as gray). Primer and probe sequences are listed in Supplementary Table 1.