A role for barley CRYPTOCHROME1 in light regulation of grain dormancy and germination

Abstract

It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g., light and cold) might direct such expression in grains. Our previous studies indicated that light is a key environmental signal inhibiting germination in dormant grains of barley (Hordeum vulgare), wheat (Triticum aestivum), and Brachypodium distachyon and that this effect attenuates as after-ripening progresses further. We found that the blue component of the light spectrum inhibits completion of germination in barley by inducing the expression of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase and dampening expression of ABA 8'-hydroxylase, thus increasing ABA content in the grain. We have now created barley transgenic lines downregulating the genes encoding the blue light receptors CRYTOCHROME (CRY1) and CRY2. Our results demonstrate that CRY1 is the key receptor perceiving and transducing the blue light signal in dormant grains.

Figure 1.

Schematic Representation of the Barley CRY1a, CRY1b, and CRY2 Genes. Barley CRY1a and CRY1b gene sequences and the CRY2 partial length gene sequence were retrieved from the National Center for Biotechnology Information and used to design RNAi cassettes (see Methods). CRY1a (A), CRY1b (B), and CRY2 (C) sequences were annotated to reveal the UTRs and coding regions. (A) and (B) A 355-bp region (red bar) containing portions of the second and third exons of CRY1a (A) and sharing 78% identity with CRY1b (B) over the same region (red bar) was chosen for silencing CRY1a/b. (C) For silencing CRY2, a 339-nucleotide region spanning the last intron in the coding sequence of that gene (red bar) and showing low similarity to CR1a/b was chosen. The 5′ UTR and 3′ UTRs (when known) are colored light blue, the coding regions of the genes are dark blue, and these are joined by chevrons depicting the introns. The translational start site (when known) is indicated by a bent arrow at the ATG, while the stop codon is denoted with a stop sign. The positions of the primers used for expression analysis are also indicated.

Figure 2.

Silencing Effects in CRY1a/b and CRY2 Transgenic Lines and Their Dormancy. (A) and (B) Expression of CRY1a/b and CRY2 in barley grains imbibed 18 h in darkness from four independent, homozygous, single-insertion CRY1a/b lines (A), four independent, homozygous, single-insertion CRY2 lines (B), and from their corresponding null segregant siblings was tested by qRT-PCR, revealing a consistent expression reduction of the targeted genes. (C) to (F) Grains from the same harvest were tested for dormancy in darkness ([C] and [D]) or under WL ([E] and [F]). Each point is the average ± se of three biological replicates. Statistically significant differences using Dunnett’s test at α = 0.05 are indicated with an asterisk.

Figure 3.

After-Ripening Time Course of the RNAi CRY1a/b and CRY2 Lines. The four lines carrying the CRY1a/b RNAi construct and the four lines carrying the CRY2 RNAi construct, together with their control null segregants, were after-ripened for various durations before being tested for germination in either continuous WL (open symbols) or darkness (filled symbols). Each point is the average ± se of final percentage germination values after 5 d from three biological replicates. Statistically significant differences between RNAi lines and null segregants using Dunnett’s test at α = 0.05 are indicated with an asterisk.

Figure 4.

CRY1a/b RNAi Lines Show a Germination Phenotype in Response to BL. Grains of CRY1a/b RNAi line 15 and its null segregant were tested for dormancy in various light qualities following partial after-ripening at 37°C. (A) Grains were germinated in darkness or under constant R, FR, BL, or WL. (B) CRY1a/b RNAi line 15 and null segregant grains were placed under constant BL at various fluence rates. Percentage of germination after 5 d was recorded.

Figure 5.

Relative Expression of Genes Involved in the BL Signaling Pathway in the CRY1a/b RNAi and Null Segregant Embryos. Reverse-transcribed mRNA from embryos from partially after-ripened grains imbibed in darkness or under BL for different durations was tested using gene-specific primers for transcript abundance of ELIP90 (A), ELIP58 (B), HY5 (C), or CIP8 (D). Each point is the average ± se of relative transcript abundances from three biological replicates of 15 embryos each. Statistically significant differences between RNAi lines and null segregants using Dunnett’s two-tailed test at α = 0.05 are indicated with an asterisk.

Figure 6.

Relative Expression of Genes Involved in ABA Metabolism in the CRY1a/b RNAi and Null Segregant Embryos. Reverse-transcribed mRNA from embryos from partially after-ripened grains imbibed in darkness or under BL for different durations was tested using gene-specific primers for transcript abundance of NCED1 (A), NCED2 (B), and ABA8’OH1 (C). Each point is the average ± se of relative transcript abundances from three biological replicates of 15 embryos each. Statistically significant differences between RNAi lines and null segregants using Dunnett’s two-tailed test at α = 0.05 are indicated with an asterisk.

Figure 7.

ABA Quantification on Embryos from the HvCRY1a/b RNAi Grains and Null Segregant Grains. ABA was extracted from embryos from dry and imbibed partially after-ripened grains. Imbibition occurred in darkness (A) or under BL (B). ABA was quantified using liquid chromatography–tandem mass spectrometry. Numbers above columns indicate the average ABA concentration for each sample. Subpanels ([a] and [b]) are shown to magnify the latest time points with a rescaled axis. Each point is the average ± se of hormone amounts from four biological replicates. Statistically significant differences between RNAi lines and null segregants using Dunnett’s test at α = 0.05 are indicated with an asterisk.

Figure 8.

A Model of the Light Regulation of Germination in the Grasses and Arabidopsis. In grasses BL, FR, and R wavelengths are able to regulate germination in dormant grains. In Arabidopsis, R and FR wavelengths have a key role in regulating germination, and a PHYB-mediated BL effect has been also detected, although no relationship with seed dormancy has been reported. Black-dotted line: In wild grasses such as B. distachyon, strong effects of R (promoting germination) and of FR and BL (inhibiting germination) are detectable in dormant grains. Blue-dotted line: In domesticated grasses such as wheat and barley, the existence of R and FR effects has not been reported, while the BL inhibitory effect is conserved. Red-dotted line: In Arabidopsis and other dicots, R and FR had a reversible effect on seed germination driven by changes in the PHYB conformation (Pfr and Pr forms), and BL can partially mimic the FR effect. In cereal grains, BL is perceived by CRY1, and possibly also by other photoreceptors such as PHYB, inducing the expression of the NCED1 gene, while repressing ABA8’OH-1, which stabilizes the ABA content in the embryo, blocking germination. FR and R wavelengths are perceived by PHYB and by other PHY family members. In grasses, the targets of R and FR are not known, but in Arabidopsis, FR induces the expression of NCED6 and NCED9, while R blocks their expression, increasing or decreasing ABA levels, respectively.