Phytochrome E Plays a Role in the Suppression of Germination in Far-Red Light in Tomato

Abstract

As photoautotrophs, plants use light not only as a source of energy but also as cues for directing growth and development. Phytochromes comprise a small gene family of plant specific light receptors that absorb mostly in the red/far-red portion of the electromagnetic spectrum. These light receptors are well-studied in the model species Arabidopsis thaliana, yet much less is known about their functions in other species. We have generated CRISPR-induced mutations in SlPHYTOCHROME E (SlPHYE) and SlPHYF, produced higher order mutants, and characterized some of their physiological functions in tomato (Solanum lycopersicum). We report that SlphyE plays a major role in detecting far-red light, repressing germination when light conditions are unfavorable for establishing a new seedling. While SlphyE functions on its own, it also synergistically works with another phytochrome, SlphyB1, which by itself only plays a minor role in germination control. Aside from its role in far-red light detection, SlPhyE is also involved in perceiving red light, leading to the repression of hypocotyl elongation and the promotion of light avoidance growth in the roots. SlPhyF acts synergistically with phyB1 during photomorphogenesis but it is not involved in far-red light detection during germination.

Keywords: CRISPR; germination; photomorphogenesis; phytochrome.

FIGURE 1

CRISPR‐induced phyE mutations create likely loss of function alleles. (A) PhyE gene model indicating exons (blue) and introns (gray), translational start (green) and stop (red) codons, and overall transcript length including 5′ and 3′ ends (thin, dark gray line). CRISPR was used to generate two alleles. (B) Nucleotide‐level detail of allele phyE‐5. C: Nucleotide‐level detail of allele phyE‐8. Sequences include: 5′ untranslated region (white background, nt 1–347), the beginning of exon 1 (starting at nt 348), start codon (nt 348–350, same green background as in A), left and right sequencing primers (light blue), PAM sequences (yellow), the two guide RNAs (orange background) and the mutated sequences in magenta with white lettering. The phyE‐5 allele has a single base pair deletion at nt 681, while the phyE‐8 mutation has a deletion of 75 bp from nt 622–696. Primer sequences can also be found in Table S1.

FIGURE 2

Phenotypes of 4‐week‐old phytochrome mutants. Seedlings were grown in 10 × 10 cm pots in potting soil without additional fertilizer. Scale: pots are 10 cm in width. Plants were grown in triplicate in the greenhouse and one representative individual is shown.

FIGURE 3

Both phyB1 and phyE play critical roles in the repression of hypocotyl elongation in R. Seedlings were germinated for 4 days in the dark and then grown for an additional 3 days either in darkness or R. Three‐way ANOVA (type I) followed by Tukey analysis was performed using the software package R. Genotypes/treatments that are not connected by the same letter are statistically significantly different from each other. The box plot shows the 25–75 percentile values between the bottom and the top of the box. The whiskers extend to maximally 1.5* IQR (inter quartile range). The bold line indicates the median. N = 1674 total.

FIGURE 4

Phytochrome E plays a critical role in preventing germination in FR light. Batches of seeds were allowed to germinate in FR light either on 500 nM ABA‐supplemented or non‐supplemented MS agar (see Section 2 for details). Three‐way ANOVA (type I) followed by Tukey analysis was performed using the software package R. Genotypes/treatments that are not connected by the same letter are statistically significantly different from each other. Each data point (N = 350) represents one batch of 15 seeds. Box plot parameters are the same as in Figure 3.

FIGURE 5

The light signal for roots to elongate is mostly mediated by phyB1, with phyE, and phyF playing minor roles. Nine‐day‐old seedlings were grown on agar plates, and either exposed to continuous R or darkness. Three‐way ANOVA (Type I) followed by Tukey analysis was performed using the software package R. Genotypes/treatments that are not connected by the same letter are statistically significantly different from each other. N = 1118 total. Box plot parameters are the same as in Figure 3.

FIGURE 6

Phytochrome E together with phyB1 mediates the light‐induced root growth response. Nine‐day‐old seedlings were either grown in R or darkness on MS medium either supplemented with 500 Sq nM ABA or grown on non‐supplemented medium. Three‐way ANOVA (Type I) followed by Tukey analysis was performed using the software package R. Genotypes/treatments that are not connected by the same letter are statistically significantly different from each other. N = 955 total. Box plot parameters are as in Figure 3. Black: no ABA in darkness, red: no ABA in R, gray: ABA in darkness, dark red: ABA in R.

FIGURE 7

Model illustrating how light quality effects the role of PhyB1/E in coordination with ABA to fine‐tune growth and developmental responses in tomato.