Tomato phyE Is Required for Shade Avoidance in the Absence of phyB1 and phyB2

Abstract

The phytochrome (phy) family of red and far-red photoreceptors provides plants with critical information about their surrounding environment and can signal downstream developmental and physiological changes. Neighboring plants compete for limited light resources, and their presence is detected by the phytochrome photoreceptors as a reduced ratio of red: far-red light. One common response to shade is increased elongation of petioles and internodes to compete with their neighbors. While the phytochrome family, phyB in particular, has been well studied in Arabidopsis, information about the other phytochrome family members is limited, especially in sympodial crop plants such as tomato, that have a very different architecture from that of the model plant. To study the tomato phytochrome family we took advantage of several existing mutants and generated an artificial miRNA (amiRNA) line to target SlPHYE, the remaining phytochrome B subfamily member with no currently available mutant line. Here, we characterize internode elongation and shade avoidance phenotypes of the SlPHYE amiRNA line (PHYE amiRNA). In addition, higher order phytochrome subfamily B mutants were generated with the PHYE amiRNA line to investigate the role of SlphyE within the phyB subfamily. We find that the PHYE amiRNA line has no detectable phenotype on its own, however in higher order combinations with SlphyB1 and/or SlphyB2 there are notable defects in shade avoidance. Most notably, we find that the triple mutant combination of SlPHYE amiRNA, SlphyB1, and SlphyB2 has a phenotype that is much stronger than the SlphyB1 SlphyB2 double, showing constitutive shade avoidance and little to no response to shade. This indicates that SlphyE is required for the shade avoidance response in the absence of SlphyB1 and SlphyB2.

Keywords: Solanum lycopersicum; elongation; internode; phytochrome; shade avoidance; tomato.

Figure 1

Phylogenetic tree of the phytochrome families of tomato, Arabidopsis, rice, grape, and Medicago truncatula.

Figure 2

Growth phenotypes of Arabidopsis and tomato phyB paralog double mutants. Hypocotyl lengths of (A) Arabidopsis phyB/phyD double mutants and wild-type Landsberg and (B) tomato phyB1/phyB2 double mutants and wild-type Moneymaker. Measurements were made 10 days post plating on seedlings grown on 0.5x MSMO and 0.7% agar in Phytatrays under constant light in high R:FR and low R:FR conditions. n = an average of 20 for each genotype/treatment. Internode length (C) of 5 week old phyB1/phyB2 and Moneymaker plants grown on soil in four-inch pots under long day (16:8) high R:FR and low R:FR conditions in a growth chamber. n = 5 plants for each genotype/treatment. The error bars show ±SE. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, NS = not significant by Student's t-test.

Figure 3

Gene expression of PHYB subfamily members (A) PHYE, (B) PHYB1, and (C) PHYB2 in Moneymaker and the PHYE amiRNA transgenic line. Leaf tissue was collected from 3-week old transgenic PHYE amiRNA and wild type Moneymaker plants grown on soil in four-inch pots under long day (16:8) high R:FR conditions in a growth chamber. n = 4 biological replicates, each consisting of three individuals for each genotype. The error bars show ±SE. ^***P < 0.005, NS = not significant by Student's t-test.

Figure 4

Growth characteristics of phyB family single mutants and amiRNA in high R:FR and in low R:FR. Combined epicotyl, internode 1, and internode 2 length of 3, 4, and 5 week-old phyB1, phyB2, PHYE amiRNA, and Moneymaker plants grown on soil in four-inch pots under long day (16:8) high R:FR and low R:FR conditions. n = 6 for each genotype/treatment. The error bars show ±SE. phyE was not significantly different than wild type in high R:FR or low R:FR at any time point (P > 0.25). Asterisks for “high R:FR” bars indicate that a mutant was significantly different than Moneymaker in high R:FR at that time. Asterisks for “low R:FR” bars indicated that a mutant had a significantly different response to shade as compared to wild type at that time. Asterisks for Moneymaker “low R:FR” are for the high R:FR vs. low R:FR comparison Moneymaker for that time point. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by a linear mixed-effects model.

Figure 5

Growth phenotypes of 5-week old high R:FR grown Moneymaker, single mutants phyA, phyB1, phyB2, PHYE amiRNA, double mutants phyB1/phyB2, phyB1/PHYE amiRNA, phyB2/PHYE amiRNA, triple mutant phyB1/phyB2/PHYE amiRNA, and quadruple mutant phyA/phyB1/phyB2/PHYE amiRNA.

Figure 6

Growth phenotypes of higher order phytochrome mutants. Total length of epicotyl, internode 1, and internode 2 of 5-week old Moneymaker and phytochrome single, double triple, and quadruple mutant plants grown on soil in four-inch pots under long day (16:8) high R:FR and low R:FR conditions in a growth chamber. n = an average of eight plants for each genotype/treatment/replicate. The error bars show ±SE. Asterisks for mutant “high R:FR” bars indicate that a mutant was significantly different than Moneymaker in high R:FR. Asterisks for mutant “low R:FR” bars indicated that a mutant had a significantly different response to shade as compared to wild type at that time. Asterisks for Moneymaker “low R:FR” are for the high R:FR vs. low R:FR comparison in Moneymaker. P-values for additional contrasts are provided in Table 3. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by a linear mixed-effects model.