3. Results
3.4 EIN2 is a potential in-vivo substrate for PRT1
3.4.5 Cytokinin-induced inhibition of hypocotyl elongation in the dark is impaired in prt1-1 ts
Next to its crucial function in ethylene signaling, considerable hormonal crosstalk converges around the axis of the EIN2 protein. Resistance to root growth inhibition by cytokinin is one of the most prominent phenotypes caused by an ein2 null allele. It was considered that PRT1 targeting EIN2 might play a role in this pathway, rather than during the triple response to ACC.
To check the role of PRT1 in cytokinin signaling, inhibition of shoot and root growth by BA was assayed. However, no difference was observed concerning size, root length, or greening of plantlets upon BA between the WT and prt1-1ts (Fig. 37), or prt1-1 homozygous backcross lines that produced siliques at 28°C (Fig. S22). In contrast, both the ein2-1 and the ein2-5 alleles conferred near insensitivity of root growth to 0.1 µM BA in the medium, and root growth of the ein2 mutant lines was also much less affected than WT or prt1-1ts seedlings in the presence of 0.5 µM BA (Fig. 37).
Next, to test if prt1 mutation affected cytokinin sensitivity in the dark, the triple-response-like effect of BA on etiolated seedlings was assessed. For these and all further experiments, the ein2-1 allele was used for comparison instead of ein2-5/DR5::GUS, since it lacked the reporter construct.
Moreover, the ein2-5 allele leads to a frameshift between the eighth and ninth transmembrane helix of the N-terminal NRAMP-like domain of EIN2, whereas ein2-1 introduces a stop codon in the hydrophilic part well behind the NRAMP-like domain, after amino acid number 555 (Alonso et al., 1999). Hence, the ein2-1 mutant line potentially still contains a truncated version of EIN2 comprising the N-terminal NRAMP-like domain which was shown to contribute to some aspects of EIN2 signaling, and is certainly required for responses upstream of CEND signaling (see introduction).
Fig. 37| Shoot and root inhibition of prt1-1ts seedlings by cytokinin. Seeds of the indicated genotypes were placed on sugar-supplemented agar medium containing 0.1 µM BA (cytokinin), 0.5 µM BA, or a mock solution (0.006% dimethyl sulfoxide (DMSO)). After stratification for 5 days, plates were transferred to 22°C and the plants were grown for 8 days under a long-day light regime (16/8). Pictures were taken of representative seedlings derived from one out of four independent experiments, each comprising at least 12 plants per genotype and growth condition. Note: The ein2-5 genotype refers to the line harboring also the DR5::GUS construct (NASC ID: N16707).
Given that PRT1-function would potentially affect only the C-terminal part of EIN2, the ein2-1 line was considered the better comparison for these experiments. For both ein2-1 and ein2-5, full germination capacity could only be reached upon extensive seed after-ripening for several months.
This effect of ethylene mutants was described before (Bleecker et al., 1988; Beaudoin et al., 2000;
Bentsink and Koornneef, 2008). However, in contrast to previous reports (Beaudoin et al., 2000), in my hands stratification did not break the prolonged seed dormancy of freshly matured ein2 seeds.
To test for their capacity for induction of the triple response to cytokinin, plants of the WT, prt1-1ts, ein2-1, and double mutant prt1-1 ein2-1 (hereafter referred to as prt1 ein2) genotypes where subjected to different concentrations of BA during germination in the dark.
Interestingly, prt1-1ts showed a pronounced loss of sensitivity towards BA treatment in the triple response assay as compared to the WT (Fig. 38). In fact, prt1-1ts lines exhibited up to 30 percent longer hypocotyls than the WT when etiolating on medium containing BA. Next to hypocotyl growth, root inhibition by cytokinin also appeared to be slightly reduced in prt1-1ts lines, but this effect was much less evident (Fig. 38A). Concerning hypocotyl length, the strongest effect was observed upon an intermediate BA concentration of 1 µM. Under these conditions, the mean hypocotyl lengths of prt1-1ts were about 90 percent of those of ein2-1, while the difference between prt1-1ts and ein2-1 got stronger upon higher levels of BA. No difference was measurable between ein2-1 and the prt1 ein2 double mutant, although the ein2-1 mutation did not confer full insensitivity to BA. Moreover, cotyledon opening was occasionally observed in the prt1-1ts line upon BA treatment (Fig. 38A).
Induction of cotyledon opening in the dark, particularly in the presence of BA, is characteristic of ethylene-insensitive mutants, since WT plants require much higher BA levels to induce cotyledon opening (Chory et al., 1994; Wei et al., 1994; Hansen et al., 2009; Cortleven et al., 2018).
The prt1-1ts line was previously found to exhibit sensitivity to elevated temperatures caused by the genomic background of the line. To address the possibility that the BA-related phenotype was also induced by the second locus, the line N119 was subjected to triple response assays. This line also contains the prt1-1 allele, but lacks the temperature-dependent sterility trait (Fig. 8B).
A
B
Fig. 38| The prt1-1 mutant displays decreased sensitivity towards cytokinin during etiolation. Seeds of the indicated genotypes were transferred to sucrose-supplemented agar plates containing different concentrations of BA or a mock additive (0.03% DMSO), chilled for 5 days in the dark, subjected to illuminated conditions at 22°C for 9 hours and subsequently kept in the dark. Hypocotyl lengths were measured after 87 hours of dark growth. A, Pictures show representative seedlings at different concentrations of BA. The arrowhead indicates a seedling of prt1-1ts with open hypocotyls. B, Quantifications of hypocotyl lengths of all individuals from the experiment exemplified in A. n≥ 30; whiskers indicate standard deviations. For statistical analysis, each treatment group was tested individually: Shapiro-Wilk normality tests, followed by one-way ANOVA and Tukey HSD; p≤ 0.001.
While the effect of ACC on N119 during etiolation was minor, like previously observed for prt1-1ts, hypocotyl lengths of N119 were markedly increased in comparison to the WT upon 1 µM BA (Fig. 39).
Hypocotyls of N119 in the presence of 1 µM BA were at least 25 percent longer than WT, resembling the effect detected for prt1-1ts. This indicated that the higher resistance to BA treatment during etiolation in prt1-1 lines was likely not accounted for by the genetic locus that triggered male sterility at elevated temperature.
Fig. 39| Inhibition of hypocotyl elongation by BA, but not by ACC, is also impaired in the prt1-1 line N119.
Seedlings of the WT, the 28°C-fertile prt1-1 line N119, ein2-1, and the double mutant prt1 ein2 were placed on media containing 1 µM BA and mock (0.001% ethanol), 1 µM ACC and mock (0.006% DMSO), or mock supplements only (0.001% ethanol and 0.006% DMSO). After 5 days stratification, plates were transferred to 22°C and exposed to light for 8 hours before etiolation for another 88 hours. A, Representative seedlings were aligned for photographing. B, Quantification of hypocotyl lengths of all seedlings from the experiment
exemplified in A. n≥ 32; whiskers indicate standard deviations. For statistical analysis, each treatment group was tested individually: Shapiro-Wilk normality tests, followed by one-way ANOVA and Tukey HSD; p≤ 0.001.
Since interplay between ethylene, cytokinin, and sugar signaling had been reported (Gibson et al., 2001; Yanagisawa et al., 2003; Price et al., 2004; Franco-Zorrilla et al., 2005; Hartig and Beck, 2006;
Laxmi et al., 2006; Kwon et al., 2011; Das et al., 2012), it was asked whether the supplementation with exogenous sucrose during heterotrophic growth in the dark contributed to the BA hyposensitive phenotype of the prt1-1 mutant seedlings. In fact, in the absence of sucrose in the medium, inhibition of hypocotyl elongation by BA was much stronger than previously observed upon sugar supplementation in N119 (Fig. 40), as well as prt1-1ts (Fig. S23), and approached WT levels. In the presence of additional sucrose, hypocotyl lengths of N119 were decreased by about 35 percent by 1 µM BA compared to mock treatment (Fig. 39, 40). In contrast, without sugar supplement, the hypocotyl elongation of N119 plants was decreased by about 50 percent (Fig. 40). WT plants, by comparison, showed a reduction of hypocotyl elongation by about 55 percent, independently of whether sugar was added to the medium or not. The BA sensitivity of ein2-1 was also marginally influenced by sucrose supplementation, with 28 and 32 percent inhibition of hypocotyl elongation on sucrose-supplemented or basal medium, respectively (Fig. 40). Thus, exogenous sucrose provided by the growth medium apparently counteracted the effect of BA in N199 and prt1-1ts mutant plants.
Fig. 40| BA-resistance of hypocotyl repression in the prt1-1 mutant line N119 is sugar-dependent. The triple response to BA was assayed with or without sugar supplementation of the medium using seedlings of WT, the prt1-1 genotype N119, and ein2-1. Seeds were placed on agar-containing plates with BA (1 µM) or mock (0.006% DMSO) addition, and 0.5 % sucrose or without sugar addition. After a 5 day period of dark stratification, seeds were illuminated at 22°C for 9 hours and subsequently incubated in the dark for additional 87 hours. n≥ 29; whiskers indicate standard deviations. For statistical analysis, each treatment group was tested individually: Shapiro-Wilk normality tests, followed by one-way ANOVA and Tukey HSD; p≤ 0.001.
Of note, in contrast to what was described previously (Zhang et al., 2010; Kushwah and Laxmi, 2014), hypocotyl-elongation under control conditions was not promoted considerably by the presence of 0.5% sucrose in the growth medium in any of the lines tested. This was potentially due to the experimental setup, since previous assays evaluated seedlings at the age of 8 days (Zhang et al., 2010; Kushwah and Laxmi, 2014), while hypocotyls were assessed at the fourth day after stratification during this work.
3.4.5.1 Cytokinin hyposensitivity is not caused by the prt1-1 allele
Hyposensitivity towards cytokinin in the triple response assay was observed in this work for two related lines carrying the prt1-1 allele, namely N119 and prt1-1ts. In order to test whether PRT1 was required for the cytokinin response, transgenic lines expressing PRT1 under control of a PRT1 promoter construct in the prt1-1ts genomic background (compare to Fig. 19) were subjected to the triple response assay.
Fig. 41| Expression of an N-terminally tagged PRT1 coding sequence does not complement the BA hyposensitivity of prt1-1ts in the triple response assay. Seeds were transferred to sucrose-supplemented agar plates containing 1 µM BA or mock (0.006% DMSO). Triple response was assayed after 5 days stratification, 8 hours light exposure at 22°C and subsequent 87 hours dark incubation of the vertical plates. Representative seedlings were aligned for photography. A, Next to WT and prt1-1ts, T2 (line 15) or T3 (all others) lines expressing N-terminally 3HA-tagged PRT1 under control of the endogenous promoter (proPRT1∆CTCT) in the prt1-1ts mutant background were assayed. Asterisks indicate lines with single insertions of the T-DNA. B, T3 lines in the prt1-1ts background carrying N-terminally GFP-tagged variants of the PRT1 coding sequence were subjected to the triple response assay. Line WT-8 contained PRT1 as the WT sequence, whereas the other lines harbored the PRT1C29A sequence with a mutation in RING1 leading to in-vitro autoubiquitination inactivity.
When assaying T2 and T3 transgenic lines bearing either N-terminally 3HA-tagged or GFP-tagged PRT1 fusion constructs, complementation of the cytokinin hyposensitive phenotype of prt1-1ts was not observed for any of the lines (Fig. 41). This strongly indicated that the loss of PRT1 function was not causal for the phenotype, even though it cannot be excluded that the N-terminal tag interfered with PRT1’s function in this particular case. To further address this possibility, backcross lines derived from a cross between prt1-1ts (female) and Col-0 WT (male) plants were included in the analysis.
These lines, designated BC #68 and BC #85, exhibited WT-like fertility at 28°C while carrying the prt1-1 allele homozygously (data not shown).
Both tested prt1-1 backcross lines had lost the cytokinin hyposensitive phenotype of reduced hypocotyl inhibition observed for prt1-1ts and N119 in the triple response assay (Fig. 42, S23).
Opposing the progenitor lines, one of the backcross lines, BC #68, even exhibited a faint reduction of dark-induced hypocotyl growth in the presence of both sucrose and BA (Fig. 42). However, this very mild effect was not measurable to statistical significance during replication of the experiment and was not seen for the backcross line BC #85. A previously observed occasional opening of the
cotyledons in response to BA was likewise lost in the backcross lines (Fig. S23). Based on these results, it was concluded that loss of PRT1 function does not affect the sensitivity towards BA during etiolation. Notably, the cytokinin hyposensitive phenotype was not observed in the backcross line BC
#30 which was sterile at 28°C (Fig. 8) but carried the WT PRT1 allele homozygously (Fig. S23). This indicated that the two phenotypes, high temperature sterility and cytokinin hyposensitivity in the dark, were genetically independent and were caused by different alterations in the genetic background of both the prt1-1ts and N119 lines.
Fig. 42| Cytokinin hyposensitivity does not depend on the prt1-1 allele. The triple response to cytokinin (1 µM BA) in the presence or absence of sucrose was analyzed using two prt1-1 backcross lines, in comparison to the WT and ein2-1 mutant genotype. Backcross lines BC #68 and #85 were derived from a cross prt1-1ts X Col-0 and were homozygous for the prt1-1 locus, but exhibited WT-like fertility at 28°C. F4 seeds were used in the assay.
The experiment was performed as described in Fig. 40. n≥ 35; whiskers indicate standard deviations. For statistical analysis, each treatment group was tested individually: Shapiro-Wilk normality tests, followed by one-way ANOVA and Tukey HSD; p≤ 0.001.