Clock-Controlled and Cold-Induced CYCLING DOF FACTOR6 Alters Growth and Development in Arabidopsis

The circadian clock represents a critical regulatory network, which allows plants to anticipate environmental changes as inputs and promote plant survival by regulating various physiological outputs. Here, we examine the function of the clock-regulated transcription factor, CYCLING DOF FACTOR 6 (CDF6), during cold stress in Arabidopsis thaliana. We found that the clock gates CDF6 transcript accumulation in the vasculature during cold stress. CDF6 mis-expression results in an altered flowering phenotype during both ambient and cold stress. A genome-wide transcriptome analysis links CDF6 to genes associated with flowering and seed germination during cold and ambient temperatures, respectively. Analysis of key floral regulators indicates that CDF6 alters flowering during cold stress by repressing photoperiodic flowering components, FLOWERING LOCUS T (FT), CONSTANS (CO), and BROTHER OF FT (BFT). Gene ontology enrichment further suggests that CDF6 regulates circadian and developmental-associated genes. These results provide insights into how the clock-controlled CDF6 modulates plant development during moderate cold stress

Clock-Controlled and Cold-Induced CYCLING DOF FACTOR6 Alters Growth and Development in Arabidopsis

The circadian clock represents a critical regulatory network, which allows plants to anticipate environmental changes as inputs and promote plant survival by regulating various physiological outputs. Here, we examine the function of the clock-regulated transcription factor, CYCLING DOF FACTOR 6 (CDF6), during cold stress in Arabidopsis thaliana. We found that the clock gates CDF6 transcript accumulation in the vasculature during cold stress. CDF6 mis-expression results in an altered flowering phenotype during both ambient and cold stress. A genome-wide transcriptome analysis links CDF6 to genes associated with flowering and seed germination during cold and ambient temperatures, respectively. Analysis of key floral regulators indicates that CDF6 alters flowering during cold stress by repressing photoperiodic flowering components, FLOWERING LOCUS T (FT), CONSTANS (CO), and BROTHER OF FT (BFT). Gene ontology enrichment further suggests that CDF6 regulates circadian and developmental-associated genes. These results provide insights into how the clock-controlled CDF6 modulates plant development during moderate cold stress

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male‐sterility in Eucalyptus without impacts on vegetative growth

Abstract Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male‐sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated knockout of three floral genes in both FT‐expressing (early‐flowering) and non‐FT genotypes. We report male‐sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3‐like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3‐like knockouts produced virtually no pollen. In addition to male‐sterility, both erec8 and ehec3‐like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3‐like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild‐type (non‐early‐flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single‐leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss‐of‐function mutations in any of these three genes show promise as a means of inducing male‐ or complete sterility without impacting vegetative development

TCP4-dependent induction of <i>CONSTANS</i> transcription requires GIGANTEA in photoperiodic flowering in <i>Arabidopsis</i>

<div><p>Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In <i>Arabidopsis thaliana</i>, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about <i>CO</i> transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as <i>CO</i> activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the <i>CO</i> promoter. TCP4 induces <i>CO</i> expression around dusk by directly associating with the <i>CO</i> promoter <i>in vivo</i>. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces <i>CO</i> expression in a <i>GI</i>-dependent manner. The physical association of TCP4 with the <i>CO</i> promoter was reduced in the <i>gi</i> mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the <i>in vivo</i> TCP4 complex, and the <i>gi</i> mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.</p></div

GI enhances TCP4-binding to the <i>CO</i> promoter.

<p>(A) The representative protein profiles of TCP4-3F6H overexpression in WT and <i>gi-2</i> background grown in LD are shown. Histone H3 protein was used as the loading control. The numbers above the images indicate time (h) after light onset within a day. (B) The quantified results of the TCP4-3F6H protein profiles shown in (A) are obtained from 3 independent biological replicates. Significant differences are indicated by asterisks (Student’s <i>t</i>-test, <i>p<</i>0.05). Data represent means ± SEM. (C) BiFC assay results of interactions among CIN-TCP proteins are shown. The full length of TCP2, 3, 5, 10, 13, 17, and 24 fused to YFP^c were co-expressed with YFPⁿ-mTCP4 in <i>N</i>. <i>benthamiana</i> leaf epidermal cells. Scale bar, 20 μm. (D) Results of ChIP analysis using <i>35S</i>:<i>TCP4-3F6H</i> in either WT and <i>gi-2</i> backgrounds harvested at ZT16 are shown. Amplicons located in the <i>CO</i> promoter are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006856#pgen.1006856.g003" target="_blank">Fig 3D</a>. Significant differences are indicated by asterisks (Student’s <i>t</i>-test, <i>p<</i>0.05). Data represent means ± SEM (<i>n</i> = 4).</p

TCP4 physically associates with <i>CO</i> promoter and activates its transcription.

<p>(A to C) Histochemical GUS staining images from plants harboring the GUS reporter controlled by the class II <i>TCP</i> promoters. Images from whole seedlings, cotyledons, and the first set of leaves in <i>TCP3</i>:<i>GUS</i> (A), <i>TCP4</i>:<i>GUS</i> (B), and <i>TCP10</i>:<i>GUS</i> (C) plants grown in LD are shown. Scale bar, 0.5 mm. (D) A diagram of the <i>CO</i> locus and the locations of 7 amplicons used in ChIP analysis. The gray box represents 5′-UTR and the white box represents the first exon. Red boxes represent class II TCP binding sites (GGACCA). (E) Results of ChIP analysis using <i>SUC2</i>:<i>mTCP4-3F6H</i> plants against the <i>CO</i> promoter harvested at different times of day are shown. mTCP4 contains synonymous mutations in the miR319-binding site. 10-day-old plants grown in LD were harvested at Zeitgeber time (ZT) 4, 13, and 22. The <i>UBQ10</i> locus was used as a control. Significant differences from WT harvested on the same ZT are indicated by asterisks (one-tailed Student’s <i>t</i>-test, <i>p<</i>0.005). Data represent means ± SEM (<i>n</i> = 4). (F) A schematic diagram of different lengths of the <i>CO</i> promoter with the location of the TCP-binding site used in (H) is depicted. Red boxes represent TCP binding sites and a pink box represents a mutated TCP binding site (<i>TCP</i>^<i>mut</i>). (G) The representative western blot images of effectors (GFP and mTCP4) and the reference [Renilla Luc (RLuc)] in each combination analyzed in (H) and quantitative results of the effector levels (relative to RLuc levels) obtained from 3 independent biological replicates are shown. (H) The results of the luciferase reporter assay in <i>N</i>. <i>benthamiana</i> are shown. The effects of <i>TCP4</i> on firefly luciferase (Luc) activities controlled by 1,000-bp of the <i>CO</i> promoter (gray bars), 500-bp (white bars), and 500-bp with a mutation on the TCP-binding site (dark gray bars) are tested. The activities of firefly Luc were normalized by the activities of RLuc. Asterisks denote significant differences in each combination (Bonferroni-corrected student’s <i>t</i>-test, <i>p<</i>0.05). Data represent means ± SEM (<i>n</i> = 3).</p

Class II CIN-TCP proteins redundantly function as transcriptional activators of <i>CO</i>.

<p>(A and B) Flowering phenotypes of single, double (A), and higher order <i>tcp</i> mutants (B) in LD were analyzed. Total number of rosette leaves and cauline leaves generated from the main stem were counted when plants bolted. Significant differences are indicated by asterisks (HSD test; *<i>p</i><0.05, **<i>p<</i>0.01, ***<i>p<</i>0.001). Data represent means ± SEM (<i>n</i>≥16). (C to F) Gene expression patterns of <i>CO</i> (C and D) and <i>FT</i> (E and F) in LD in single, double (C and E), and higher order <i>tcp</i> mutants (D and F) are shown. Significant differences from WT values are indicated by asterisks (<i>p<</i>0.05, Dunnett’s test). Data represent means ± SEM (<i>n</i> = 3).</p

TCP and FBH coordinately regulate <i>CO</i> expression.

<p>(A to C, E and F) Gene expression patterns of <i>CO</i> (A and E), <i>FT</i> (B and F), and <i>FBH1</i> (C) in <i>fbh tcp</i> combinational mutants (A and B) and <i>35S</i>:<i>FBH1</i> with or without higher order <i>tcp</i> mutation (C, E and F) grown in LD are shown. For (A) and (B), significant differences from WT values are indicated by asterisks (<i>p<</i>0.05, Dunnet’s test). (D) The flowering phenotype of the <i>35S</i>:<i>FBH1</i> plants grown in LD. Total number of rosette leaves and cauline leaves generated from the main stem were counted when plants bolted. ns: no significance (HSD test). Data represent means ± SEM (<i>n</i>≥15). For (C), (E) and (F), significant differences between the <i>35S</i>:<i>FBH1</i> lines and their background strains are indicated by asterisks (<i>p<</i>0.05, Student’s <i>t</i>-test). Data represent means ± SEM (<i>n</i> = 3).</p

TCP4 physically interacts with GI.

<p>(A) Protein-protein interaction between TCP4 and GI was analyzed in yeast. Full-length TCP4 and full-length or truncated GI fused to the DNA-binding domain (DBD) or the activation domain (AD) of Gal4 were tested under selective [–LWH (top),–LWH+10 mM 3-aminotriazole (3-AT, middle)] and non-selective [–LW (bottom)] conditions. For GI, N and C indicate the amino acid residues 1–391 and 382–1173, respectively. (B) Subcellular localization of TCP4 fused to enhanced YFP (TCP4-YFP) was observed in <i>N</i>. <i>benthamiana</i> epidermal cells. H2B-RFP was used for the nuclear marker. Images from YFP and RFP channels were merged with bright-field (BF) images. (C) BiFC assays of interaction between mTCP4 and GI are shown. The full-length of mTCP4 fused to the N-terminal half of enhanced YFP (YFPⁿ-mTCP4) and the full-length of GI fused to the C-terminal half of enhanced YFP (YFP^c-GI) were co-expressed in <i>N</i>. <i>benthamiana</i> leaf epidermal cells. Nuclear-localized form of GST fragment (GST_NLS) fused to YFPⁿ or YFP^c was used as negative control. Scale bar, 20 μm. (D) Co-immunoprecipitation (Co-IP) assay of HA-mTCP4 and GI-3F6H was performed. Proteins were expressed in <i>N</i>. <i>benthamiana</i>.</p