NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana

We thank Dr. Ben Smith (University of Oklahoma) for assistance with FLIM-FRET measurements and Dr. Min Ni (University of Minnesota) for critical reading of the manuscript. The cop1-4 mutant allele and cop1-4 co-9 cross were kindly provided by George Coupland (Max Planck Institute).Author Summary Light perception is critically important for the fitness of plants in both natural and agricultural settings. Plants not only use light for photosynthesis, but also as a cue for proper development. As a seedling emerges from soil it must determine the light environment and adopt an appropriate growth habit. When blue and red wavelengths are the dominant sources of light, plants will undergo photomorphogenesis. Photomorphogenesis describes a number of developmental responses initiated by light in a seedling, and includes shortened stems and establishing the ability to photosynthesize. The genes regulating photomorphogenesis have been studied extensively, but a complete picture remains elusive. Here we describe the finding that NUCLEAR FACTOR-Y (NF-Y) genes are positive regulators of photomorphogenesis—i.e., in plants where NF-Y genes are mutated, they display some characteristics of dark grown plants, even though they are in the light. Our data suggests that the roles of NF-Y genes in light perception do not fit in easily with those of other described pathways. Thus, studying these genes promises to help develop a more complete picture of how light drives plant development.Yeshttp://www.plosgenetics.org/static/editorial#pee

Die Rolle von Cytokinin in der lichtabhängigen Keimung von Arabidopsis thaliana

Seed germination is a precisely controlled process, involving multiple regulatory pathways. Cytokinin (CK) has been proposed to negatively regulate light-dependent seed germination in A. thaliana. Thus, the overall aim of the current study was to provide an in-depth analysis of the role of CK in seed germination induced by red (R) and far-red (FR) light. In this work, the seed germination phenotype of various CK mutant- and transgenic plants in low and very low fluence R and FR light conditions was studied. Germination rates of CK mutants and transgenic lines were significantly higher than wild-type germination rates, underpinning the negative impact of CK on seed germination in low fluence R light as well as in very low fluence FR light. This study further identified increased germination rates compared to the wild type in seeds (i) impaired in CK biosynthesis, (ii) with an increased CK catabolism, (iii) with a reduced signal perception at the level of histidine kinase receptors (AHKs) and (iv) seeds impaired in CK signal transduction via histidine phosphotransfer proteins (AHPs). Consequently, the signaling components in the respective mutants may well contribute to the repressive effects of CK on germination. A putative role of signaling components downstream of AHPs, such as A- and B-type response regulators (ARRs) in very low fluence germination need further clarification. The photoreceptor phytochrome A (phyA) is an essential part of the regulatory pathway controlling the onset of germination in very low fluence FR light. Germination assays analyzing CK biosynthesis- and CK receptor mutants lacking functional phyA led to the conclusion that phyA is essential for the induction of germination in very low fluence FR light both in the wild type and in CK mutants. Quantification of phyA protein levels did not confirm a repressive effect of CK on phyA abundance in seeds. To dissect the hormonal pathways which may influence germination of CK mutant seeds in very low fluence FR light conditions, the contribution of abscisic acid (ABA) and gibberellins (GA) was analyzed into more detail. Hormone measurements indicated neither elevated GA level nor decreased ABA level in CK deficient seeds, suggesting a CK-independent regulation of bioactive GA- and ABA levels in imbibed seeds. Interestingly, CK negatively influenced GA sensitivity, which may be an additional mechanism for CK to suppress germination. However, CK had no measurable effect on ABA sensitivity. A multitude of maternal effects are known to shape the germination response of the offspring. The present thesis revealed, that a lower CK status in maternal seed tissues led to increased germination rates of the respective seeds in FR light. However, a reduction of the CK status exclusively in the testa or the endosperm was not sufficient to increase germination rates significantly in FR light compared to wild-type seeds. These results exemplify the prominent role of CK as a negative regulator of germination in seed tissues with a higher maternal genome dosage. In the present thesis, also the gene regulatory network underlying the negative effects of CK on the germination processes in FR light was studied. Although the seeds' CK status had only a minor effect on transcriptomic changes during imbibition, a major reprogramming of the transcriptome during FR light-induced germination dependent on the seeds' CK status was evident.   Overrepresented GO categories revealed that lipid-associated, seed maturation-associated and cell wall organization-associated transcripts were differentially regulated in ahk2 ahk3 seeds in response to FR light. These results indicate that the aforementioned pathways might be relevant for the negative impact of CK on seed germination in very low fluence light. Additionally, environmental factors such as the light environment of parental plants during seed development affect the germination phenotype of their offspring. This thesis demonstrated, that growth of parental plants in shade light conditions (enriched in FR light) did not affect the germination response of their F1 offspring in FR light. However, when parental plants were grown for two subsequent generations in shaded conditions, germination rates of F2 seeds were increased in very low fluence FR light conditions. These effects were independent of the seeds' CK status. Since CK is a prominent regulator of seed size, in the last part of this work the connection between CK, seed size and seed age in FR light-induced germination was investigated. The current study found smaller seeds to germinate better when germination was induced by FR light, again this effect was similar in wild-type seeds and seeds with a reduced CK signal transduction. Regarding seed age, the repressive effect of CK on germination in FR light was retained in aged seeds during long-term storage. Overall, the presented results improve the understanding how seed germination in non-optimal low and very low fluence light conditions is regulated by CK. CK exerts a negative influence on germination, which is not dependent on altered GA or ABA hormone levels, but seems to involve seed tissues with a higher maternal genome dosage

<i>NF-YC3</i>, <i>4</i>, and <i>9</i> contribute redundantly to suppression of hypocotyl elongation in white light.

<p>Hypocotyl lengths are shown for plants grown for five days on B5 media in <b>A)</b> cWL, <b>B-C)</b> SD, or <b>D)</b> cD conditions. No differences were detected at earlier time points in cD-grown plants. Statistically significant differences (or lack thereof) are represented by lettering above bars (error bars represent 95% confidence intervals). Statistical differences were determined by ANOVA (P<0.01) and subsequent multiple comparisons by either Tukey’s (cWL) or Dunnett’s (SD, cD) procedures. Scale bar in C) represents 2mm.</p

Synergistically longer hypocotyls in <i>nf-yc triple hy5</i> mutants are a function of moderately more cells and greatly increased cell elongation.

<p>Single linear files of hypocotyl epidermal cells were both <b>A)</b> counted and <b>B)</b> measured for mean length. Plant were grown in cWL and measurements were taken on five day old plants. <b>C)</b> Example hypocotyls for each genotype near the cotyledon junction—blue color marks a representative single cell in each genotype. Arrows point to typical epidermal cells for each genotype. Scale bar = 100μm.</p

FRET-FLIM analysis shows a strong NF-YC9 by HY5 physical interaction.

<p>FRET experiments were conducted in tobacco leaves through transient 35S-driven overexpression of NF-YCs tagged with mCer3 and HY5 or NF-YB2 tagged with YFP. <b>A)</b> Nuclei expressing both mCer3 and YFP constructs were assayed for FRET through both FRAP and FLIM. <b>B)</b> A FRAP curve representative of a positive FRET result between two known interacting proteins, NF-YB2 and NF-YC9. Fluorescence intensity was calculated relative to the pre-photobleached intensity of each fluorescent protein. Yellow bars represent the timing of photobleaching events. <b>C)</b> FLIM was employed to detect FRET through lifetime measurements before and after acceptor photobleaching (FRAP) within the same nucleus. Each point is an independent combination of mCer3- and YFP-tagged proteins, and represents the shift in fluorescent lifetime elicited by acceptor photobleaching. Scale bar in A) represents 5μm. Error bars in B-C) represent 95% confidence intervals with an n ≥ 3.</p

NF-YC3, 4, and 9 are necessary for suppression of hypocotyl elongation in both cB and cR light.

<p>Hypocotyl lengths are shown for five day old plants grown on B5 media in <b>A)</b> cB (38μmol m^-2 s^-1), <b>B)</b> cFR (5μmol m^-2 s^-1), and <b>C)</b> cR (6μmol m^-2 s^-1). Statistically significant differences between groups (or lack thereof) are represented by lettering above bars (error bars represent 95% confidence intervals). Statistical differences were determined by standard ANOVA (p<0.01) when variances were not significantly different (cFR and cR) and Kruskal-Wallis ANOVA (non-parametric test, p<0.05) when variances were unequal (cB). Subsequent multiple comparisons were performed by either Tukey’s (cFR, cR) or Dunn’s (cB) procedures, respectively.</p

<i>nf-yc</i> mutants have broad light perception defects that are at least partly independent of <i>HY5</i> and do not completely overlap with those of <i>HFR1</i> and <i>LAF1</i>.

<p>Fluence rate curves for hypocotyl lengths are shown for five day old plants in <b>A)</b> cB, <b>B)</b> cFR, and <b>C)</b> cR light conditions (see symbols key in A). <b>D)</b> qPCR of <i>HFR1</i>, <i>LAF1</i>,and <i>HY5</i> in key genetic backgrounds. <b>E-F)</b> Quantification and images of typical phenotypes for mutants grown in low fluence rate cR (4.8μmol m^-2 s^-1). Scale bar in F = 2mm. <b>G-H)</b> Quantification and images of typical phenotypes for mutants grown in SD, white light conditions. Scale bar in H = 2mm. Statistically significant differences were determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006333#pgen.1006333.g002" target="_blank">Fig 2</a>.</p

Multiple <i>cop1-4</i> mutant phenotypes are partially dependent on <i>NF-YC</i> genes.

<p>Partial suppression of <i>cop1</i> mutant phenotypes are quantified for <b>A)</b> dark-grown seedling hypocotyl elongation, <b>B)</b> rosette diameter, <b>C)</b> flowering time, and <b>D)</b> relative <i>FT</i> expression levels. Statistics and labeling as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006333#pgen.1006333.g002" target="_blank">Fig 2</a>, except <i>FT</i> expression statistics which were calculated using qBase software (Biogazelle).</p

Light perception is synergistically defective in <i>nf-yc triple hy5</i> mutants.

<p>Hypocotyl lengths are shown for five day old plants grown on B5 media in <b>A-B)</b> SD, <b>C)</b> cWL, and <b>D)</b> cD. Statistically significant differences were determined and described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006333#pgen.1006333.g002" target="_blank">Fig 2</a>. Scale bar in B) represents 2mm.</p