Diversity of floral regulatory genes of japonica rice cultivated at northern latitudes

Abstract

Background: Rice is considered a short day plant. Originally from tropical regions rice has been progressively adapted to temperate climates and long day conditions in part by modulating its sensitivity to day length. Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) that code for florigens, are known as major regulatory genes of floral transition in rice. Both Hd3a and RFT1 are regulated by Early heading date 1 (Ehd1) and Days to heading on chromosome 2 (DTH2) while Heading date 1 (Hd1) also governs Hd3a expression. To investigate the mechanism of rice adaptation to temperate climates we have analyzed the natural variation of these five genes in a collection of japonica rice representing the genetic diversity of long day cultivated rice.

Results: We have investigated polymorphisms of Hd3a, RFT1, Ehd1, Hd1 and DTH2 in a collection of 57 japonica varieties. Hd3a and RFT1 were highly conserved, displaying one major allele. Expression analysis suggested that RFT1 rather than Hd3a could be the pivotal gene controlling flowering under long day conditions. While few alleles were found in the Ehd1 promoter and DTH2 coding region, a high degree of variation in Hd1, including non-functional alleles, was observed. Correlation analysis between gene expression levels and flowering periods suggested the occurrence of other factors, additionally to Ehd1, affecting RFT1 regulation in long day adapted cultivars.

Conclusions: During domestication, rice expansion was accompanied by changes in the regulatory mechanism of flowering. The existence of non-functional Hd1 alleles and the lack of correlation of their presence with flowering times in plants grown under long day conditions, indicate a minor role of this branch in this process and the existence of an alternative regulatory pathway in northern latitudes. Expression analysis data and a high degree of conservation of RFT1 suggested that this gene could be the main factor regulating flowering among japonica cultivars adapted to northern areas. In the absence of inhibition exerted by Hd1 through repression of Hd3a expression, the role of Ehd1 as a regulator of RFT1 and Hd3a appears to be reinforced. Data also indicated the occurrence of additional regulatory factors controlling flowering.

Figure 1

Distribution of the mean flowering time. Frequency distribution of the mean flowering time in japonica rice core collection grown under natural long day.

Figure 2

Correlation of flowering time with RNA levels of Hd3a or RFT1. Correlation of flowering time with RNA levels of (A) RFT1 or (B) Hd3a in leaves. RNA levels were determined by real-time RT-PCR and shown as natural logarithms. The square of Pearson’s product–moment correlation coefficient is indicated (P < 0,05). Color dots indicate cultivars carrying different types of RFT1 or Hd3a alleles.

Figure 3

RFT1 and Hd3a are conserved among cultivated rice. Nucleotide polymorphism in the RFT1 promoter (A), RFT1 coding region (B) and Hd3a promoter (C). The nucleotide sequences in the cultivars were compared with those of Nipponbare (indicated as Type A). Polymorphic nucleotides are indicated in grey. Numbers indicate position from ATG start codon. Amino acid substitutions caused by nucleotide polymorphism are indicated. The number of cultivars containing each type of allele is indicated in the right column. Arrow head indicates the start of the 5’-untraslated region.

Figure 4

Nucleotide polymorphisms in Ehd1 promoter and its relationship with flowering time. (A) Nucleotide polymorphism in the Ehd1 promoter. The nucleotide sequences in the cultivars were compared with those of Nipponbare (indicated as Type A). Polymorphic nucleotides are indicated in grey. Numbers indicate position from ATG start codon. The number of cultivars containing each type of allele is indicated in the right column. (B) Correlation of flowering time with RNA levels of Ehd1 in leaves. RNA levels were determined by real-time RT-PCR and shown as natural logarithms. The square of Pearson’s product–moment correlation coefficient is indicated (P < 0,05). Color dots indicate cultivars carrying different types of Ehd1 alleles. (C)Ehd1 RNA levels in cultivars with type A, B or C Ehd1 promoters. RNA levels represented the mean and are shown as natural logarithms. Error bands represent standard deviation.

Figure 5

Variations in Hd1 and its relationship with Hd3a expression levels. (A) Nucleotide polymorphism in the Hd1 coding region. The nucleotide sequences in the cultivars were compared with those of Nipponbare (indicated as Type 1). Polymorphic nucleotides are indicated in black or grey. Amino acid substitutions caused by nucleotide polymorphism are indicated, frame shift is indicated as F.S. Polymorphisms labeled in black caused loss of function alleles. Numbers indicate position from ATG start codon. The number of cultivars containing each type of allele is indicated in the right column. Shaded boxes represent the Hd1 zinc finger domain and CCT domain. (B) Correlation of flowering time with RFT1 and Hd3a RNA levels of cultivars carrying functional (blue dots) or non-functional Hd1 alleles (red dots). RNA levels were determined by real-time RT-PCR and shown as natural logarithms.

Figure 6

Variations in DTH2 and its relationship with flowering time. (A) Nucleotide polymorphism in the DTH2 coding region. The nucleotide sequences in the cultivars were compared with those of Nipponbare (indicated as Type A). Polymorphic nucleotides are indicated in grey. Amino acid substitutions caused by nucleotide polymorphism are indicated. Numbers indicate position from ATG start codon. The number of cultivars containing each type of allele is indicated in the right column. (B) Days to flowering of the cultivars carrying DTH2 allele type A or B. Data represented the mean and the error bands the standard deviation.