Retrograde induction of phyB orchestrates ethylene-auxin hierarchy to regulate growth

Exquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here we demonstrate enhanced phytochrome B protein abundance in red-light grown MEcPP-accumulating mutant (ceh1) plant relative to wild-type seedlings. We further establish MEcPP-mediated coordination of phytochrome B with auxin and ethylene signaling pathways, and uncover differential hypocotyl growth of red-light grown seedlings in response to these phytohormones. Genetic and pharmacological interference with ethylene and auxin pathways outline the hierarchy of responses, placing auxin epistatic to the ethylene signaling pathway. Collectively, our finding establishes the key role of a plastidial retrograde metabolite in orchestrating the transduction of a repertoire of signaling cascades, and positions plastids at the zenith of relaying information coordinating external signals and internal regulatory circuitry to secure organismal integrity. Two sentence summary The plastidial retrograde metabolite, MEcPP, orchestrates coordination of light and hormonal signaling cascade through induction of phytochrome B abundance and modulation of auxin and ethylene levels for optimal adaptive responses to light environment.

numerous adaptive processes, the nature and the operational mode of action of 126 retrograde signals have remained poorly understood.
Here we have identified MEcPP as the retrograde signaling metabolite that 179 coordinates internal and external cues, and we further delineated light and hormonal 180 signaling cascades that elicit adaptive responses to ultimately drive growth-regulating 181 processes tailored to the prevailing environment. Rc, further implicate blue light receptor, cytochrome, (Yu et al., 2010) in regulating 203 growth of these seedlings ( Fig. S1A & B). Additionally, ceh1 and ceh1/phyB-9 204 seedlings grown under Bc exhibited equally shortened hypocotyls, and under FRc 205 light the growth was almost similarly retarded in all genotypes ( Fig. S1A & B). 206 Collectively, these results support involvement of cryptochrome as well and phyB in 207 ceh1 hypocotyl growth, albeit at different degrees. However, the more drastic effect of 208 phyB in regulating hypocotyl growth of Rc grown high MEcPP accumulating 209 seedlings in conjunction with the supporting evidence from the earlier data using  Next, we measured MEcPP levels in the four genotypes grown in the dark and various 213 9 monochromatic wave lengths to examine a potential correlation between growth 214 10 phenotypes and altered levels of the retrograde signaling metabolite (Fig. 1C & S1C).

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The analyses showed almost undetectable MEcPP levels in all the dark grown 216 genotypes, and low levels of the metabolite in the Rc-grown WT and phyB-9 217 seedlings. In contrast, ceh1 seedlings grown in Rc accumulated high MEcPP levels, a 218 phenotype that was partially (~10-fold) suppressed in ceh1/phyB-9 seedlings. This    show similar PIN1 transcript levels in ceh1 and the WT seedlings (Fig. 6A). In 339 contrast, the combined approaches of immunoblot and immunolocalization analyses 340 confirmed a significant reduction in PIN1 protein levels in ceh1 compared to WT 341 seedlings ( Fig. 6B-C). Specifically, immunolocalization clearly showed reduced PIN1 342 protein abundance in plasma membranes of xylem parenchyma cells (along tracheids), 343 most notably in meristems of ceh1 compared to WT seedling, albeit with an 344 unchanged polarity (Fig. 6C). These data support the earlier finding establishing the  (Table S1, and Fig. S4). The data 362 shows that compared to WT seedlings there is a prominent reduction in the transcript 363 levels of ACS4 in the dark (≥2-fold) and the Rc (~60-fold) grown ceh1 seedlings, as 364 well as a notable (3-to 10-fold depending on the gene) reduced expression of ACS5, 6, 365 8 albeit solely in Rc-grown ceh1 (Fig. 7A).

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Measurements of ethylene in these seedlings confirmed reduced levels (~80%) of the 367 hormone in Rc-grown ceh1 compared to WT seedlings (Fig. 7B). This led us to The partial recovery of ceh1 hypocotyl growth by external application of auxin and 377 21 ethylene, albeit to varying degrees, prompted us to genetically explore their potential 378 interdependency and hierarchy of their respective growth regulatory actions in Rc 379 grown seedlings. To address this, we applied ACC and IAA independently to single 380 and double mutant lines of ceh1 introgressed into auxin receptor mutant tir1-1 381 (ceh1/tir1-1), and into single and double ethylene signaling mutants ein3 and eil1 382 (ceh1/ein3, ceh1/eil1 and ceh1/ein3 eil1). 383 Analyses of hypocotyl lengths of Rc-grown WT, ceh1, ceh1/tir1-1 and tir1-1 384 seedlings in the absence and presence of ACC demonstrate TIR1-dependent growth 385 promoting action of ACC in tir1-1 and ceh1/tir1-1 (Fig. 8A-B). We furthered these 386 studies by applying ACC alone or together with NPA ( Fig. 8C-D). Consistent with the 387 earlier data, ACC treatment promoted ceh1 hypocotyl growth, but less effectively 388 when combined with auxin polar transport inhibitor, NPA (Fig. 8C-D).

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In parallel, we examined hypocotyl growth of Rc-grown WT, ceh1, ein3, ceh1/ein3,  The EdgeR Bioconductor package implemented in R was used to generate the 503 pseudo-normalized counts for visualization and to carry out differential gene 504 expression analysis (Robinson et al., 2010). Genes were kept for further analysis if 505 read counts were greater than 1 count per million (cpm) in at least 3 of the 12 libraries.

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The EdgeR Generalized linear models (GLM) framework with explanatory variables 507 of genotype and treatment allowed us to specify a design matrix estimating the effect 508 of run number (batch) as a nuisance parameter. After fitting the model for our 509 experiment, we defined contrasts between parent lines (WT) and mutant (ceh1) in red 510 light and tested for significant expression differences using a likelihood ratio test The corresponding solvents were used as control treatment (mock)  RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen).

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At4g26410 was used to normalize target gene expressions. Gene-specific primers 552 were designed using QuantPrime q-PCR primer design tool 553 (http://www.quantprime.de/) and are listed (Supplemental Table 2). Each experiment 554 was performed with three biological replicates and three technical replicates. with X-ray, and subsequently scanned with Epson Perfection V600 Photo Scanner.

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All experiments were performed with at least three biological replicates. Data are 573 mean ± standard deviation (SD). The Statistical test was performed using library 574 agricolae, Tukey's HSD test method in R with a significance of P < 0.05 (Bunn, 2008). 575 We have specified the method we used for statistical test in all figure legends. for generously providing us with the phyB antibody. We would like to thank Jacob

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North for all his efforts towards seedling preparation. We are thankful to Dr. Geoffrey

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Benn for performing the statistical analyses using R program.