Rossana Henriques

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method

Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens–mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01–0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20–30 plants per pot) within 2–3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.

PSEUDO-RESPONSE REGULATORS 9, 7, and 5 Are Transcriptional Repressors in the Arabidopsis Circadian Clock

PSEUDORESPONSE REGULATOR9 (PRR9), PRR7, and PRR5 regulate the Arabidopsis circadian clock. PRR9, PRR7, and PRR5 proteins associate with CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL promoters in a sequential manner to repress their expression. A conserved region common to the three PRR proteins is sufficient for repressor activity. An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid‐signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2‐1, regulated by the 3′‐phosphoinositide‐dependent kinase‐1 (AtPDK1). AGC2‐1 specifically interacts with AtPDK1 through a conserved C‐terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2‐1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2‐1 in an AtPDK1‐dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2‐1 is abundant in fast‐growing organs and dividing cells, and activated during re‐entry of cells into the cell cycle after sugar starvation‐induced G1‐phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1‐regulated AGC2‐1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP‐AGC2‐1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2‐1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2‐1 in root hair growth and development.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Targeted Degradation of PSEUDO-RESPONSE REGULATOR5 by an SCFZTL Complex Regulates Clock Function and Photomorphogenesis in Arabidopsis thaliana

Circadian clocks comprise several regulatory feedback loops that control gene transcription. However, recent evidence has shown that posttranslational mechanisms are also required for clock function. In Arabidopsis thaliana, members of the PSEUDO-RESPONSE REGULATOR (PRR) family were proposed to be components of the central oscillator. Using a PRR5-specific antibody, we characterized changes in PRR5 protein levels in relation to its mRNA levels under various circadian conditions. Under long-day conditions, PRR5 mRNA levels are undetectable at dusk but PRR5 protein levels remain maximal. Upon dark transition, however, PRR5 levels decrease rapidly, indicating dark-induced, posttranslational regulation. We demonstrated that the Pseudo-Receiver (PR) domain of PRR5 interacts directly with the F box protein ZEITLUPE (ZTL) in vitro and in vivo. Analyses of mutants and transgenic plants revealed an inverse correlation between PRR5 and ZTL levels, which depends on the PR domain. These results indicate that PRR5 is negatively regulated by ZTL, which likely mediates its ubiquitination and degradation. Phenotypic analyses of prr5 ztl double mutants showed that PRR5 is required for ZTL functions. ZTL contains a Light-Oxygen-Voltage domain, and its activity may be directly regulated by blue light. Consistent with this notion, we found that blue light stabilizes PRR5, although it does not alter ZTL levels. Together, our results show that ZTL targets PRR5 for degradation by 26S proteasomes in the circadian clock and in early photomorphogenesis.

Growth signalling pathways in Arabidopsis and the AGC protein kinases

Lipid-derived signals are central to regulating a multitude of cellular processes but, in plants, little is known of the downstream signalling pathways. The Arabidopsis 3-phosphoinositide-dependent protein kinase (PDK1) could couple lipid signals to the activation of several protein kinases of the so-called AGC kinase family. The Arabidopsis AGC kinases contain sequence motives required for the docking of PDK1 and phosphorylation of their activation loop in the kinase catalytic domain. It is becoming evident that specific members of the AGC kinases are implicated in key growth signalling pathways. For example, Arabidopsis p70(S6K) might be a nodal point able to integrate hormonal and developmental signals with nutritional inputs, together with the Arabidopsis Target of Rapamycin (TOR) protein.

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

This work identifies COP1 as the ubiquitin E3 ligase for not only phytochrome B but also other members of the stable phytochrome family and shows that PIF transcription factors enhance phyB ubiquitination by COP1 in vitro. It provides a molecular mechanism for the termination of red light signal transduction. Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

In Arabidopsis thaliana, the F-box protein ZTL affects the period length of the circadian clock by regulating the stability of the core clock proteins TOC1 and PRR5. This study shows that, together with ZTL, the ZTL homologs FKF1 and LKP2 are also involved in the same protein stability regulation to determine the pace and robustness of the plant circadian clock. Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes

Post‐embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB–RBR1 (retinoblastoma‐related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FAΔRB) lacking the RBR1‐binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FAΔRB and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways.

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1–E2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.