Juan Carlos del Pozo

Arabidopsis E2Fc Functions in Cell Division and Is Degraded by the Ubiquitin-SCFAtSKP2 Pathway in Response to Light

Selective ubiquitin-mediated proteolysis through the cell cycle controls the availability, and therefore the activity, of several cell proliferation proteins. E2F transcription factors play distinct roles in both proliferating and differentiated cells by regulating gene expression. Here, we report that Arabidopsis AtE2Fc is regulated by a balance between gene expression and ubiquitin-proteasome proteolysis. AtE2Fc degradation implicates the function of the E3 ubiquitin-ligase Skp1, Cullin, F-box (SCFAtSKP2) complex and seems to be dependent on cyclin-dependent kinase phosphorylation. In addition, we found that AtE2Fc degradation is triggered by light stimulation of dark-grown seedlings. Interestingly, the auxin response mutant axr1-12, in which RUB1 modification of the SCF component CUL1 is impaired, shows increased AtE2Fc protein levels, suggesting a dysfunction in the control of AtE2Fc stability. Likewise, overexpression of a stable form of the AtE2Fc protein negatively affects cell division and increases cell size. These effects are mediated, at least in part, by downregulating the cell cycle gene AtCDC6. The negative role of AtE2Fc in gene expression is further supported by the fact that AtE2Fc interacts with plant retinoblastoma-related protein, suggesting that AtE2Fc might form part of a repressor complex. We propose that AtE2Fc might play a role in cell division and during the transition from skotomorphogenesis to photomorphogenesis.

The Balance between Cell Division and Endoreplication Depends on E2FC-DPB, Transcription Factors Regulated by the Ubiquitin-SCFSKP2A Pathway in Arabidopsis

The balance between cell proliferation, cell cycle arrest, and differentiation needed to maintain the organogenetic program depends on the coordination of gene expression, posttranslational modification, and specific proteolysis of cell cycle regulators. The G1/S and G2/M transitions are critical checkpoints controlled, in part, by cyclin-dependent kinases in the retinoblastoma (RBR)/E2F/DP pathway. Arabidopsis thaliana DPB is regulated by phosphorylation and targeted to proteasome-mediated proteolysis by the SCFSKP2A complex. In addition, DPB interacts in vivo with E2FC, because ectopic coexpression of E2FC and DPB produces severe developmental defects. To understand E2FC/DPB heterodimer function, we analyzed the effect of reducing E2FC mRNA levels with RNA interference. The e2fc-R plants developed organs with more but smaller cells and showed increased cell cycle marker gene expression and increased proliferative activity in developing leaves, meristems, and pericycle cells. This last feature produces plants with more lateral roots, consistent with an E2FC role in restricting lateral root initiation. The e2fc-R plants also show marked reductions in ploidy levels of mature leaves. These results indicate that the transition from cell division to the endocycle is sensitive to different pathways, E2FC/DPB being one of them. Our results show that E2FC/DPB is a key factor in controlling the balance between cell proliferation and the switch to the endocycle program.

The Arabidopsis Cell Cycle F-Box Protein SKP2A Binds to Auxin

This work shows that auxin binds to the cell cycle F-box SKP2A to regulate its proteolysis and DPPB and E2FC degradation. Auxin seems to regulate the interaction between SKP2A and DPB. Mutant SKP2A proteins that do not bind auxin are unable to interact with DPB and to promote cell division. These findings provide evidence that SKP2A directly connects auxin and cell division. Arabidopsis thaliana S-Phase Kinase-Associated Protein 2A (SKP2A) is an F-box protein that regulates the proteolysis of cell cycle transcription factors. The plant hormone auxin regulates multiple aspects of plant growth and development, including cell division. We found that auxin induces the ubiquitin-dependent degradation of SKP2A both in vivo and in vitro, suggesting that this hormone acts as a signal to trigger SKP2A proteolysis. In this article, we show that auxin binds directly and specifically to SKP2A. By TIR1-based superposition and docking analyzes, we identified an auxin binding site in SKP2A. Mutations in this binding site reduce the ability of SKP2A to bind to auxin and generate nondegradable SKP2A forms. In addition, these non-auxin binding proteins are unable to promote E2FC/DPB degradation in vivo or to induce cell division in the root meristem. Auxin binds to TIR1 to promote its interaction with the auxin/indole-3-acetic acid target proteins. Here, we show that auxin also enhanced the interaction between SKP2A and DPB. Finally, a mutation in SKP2A leads to auxin-resistant root growth, an effect that is additive with the tir1-1 phenotype. Thus, our data indicate that SKP2A is an auxin binding protein that connects auxin signaling with cell division.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non‐host tobacco, was found to also suppress the production of defence‐associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His6‐tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1D299A non‐catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

The HVE/CAND1 gene is required for the early patterning of leaf venation in Arabidopsis.

The hemivenata-1 (hve-1) recessive allele was isolated in a search for natural variations in the leaf venation pattern of Arabidopsis thaliana, where it was seen to cause extremely simple venation in vegetative leaves and cotyledons, increased shoot branching, and reduced root waving and fertility, traits that are reminiscent of some mutants deficient in auxin signaling. Reduced sensitivity to exogenous auxin was found in the hve-1 mutant, which otherwise displayed a wild-type response to auxin transport inhibitors. The HVE gene was positionally cloned and found to encode a CAND1 protein. The hve-1 mutation caused mis-splicing of the transcripts of the HVE/CAND1 gene and a vein phenotype indistinguishable from that of hve-2 and hve-3, two putatively null T-DNA alleles. Inflorescence size and fertility were more affected by hve-2 and hve-3, suggesting that hve-1 is hypomorphic. The simple venation pattern of hve plants seems to arise from an early patterning defect. We found that HVE/CAND1 binds to CULLIN1, and that the venation patterns of axr1 and hve mutants are similar, which suggest that ubiquitin-mediated auxin signaling is required for venation patterning in laminar organs, the only exception being cauline leaves. Our analyses of double mutant and transgenic plants indicated that auxin transport and perception act independently to pattern leaf veins, and that the HVE/CAND1 gene acts upstream of ATHB-8 at least in higher order veins, in a pathway that involves AXR1, but not LOP1, PIN1, CVP1 or CVP2.

Auxin and Epigenetic Regulation of SKP2B, an F-Box That Represses Lateral Root Formation

In plants, lateral roots originate from pericycle founder cells that are specified at regular intervals along the main root. Here, we show that Arabidopsis (Arabidopsis thaliana) SKP2B (for S-Phase Kinase-Associated Protein2B), an F-box protein, negatively regulates cell cycle and lateral root formation as it represses meristematic and founder cell divisions. According to its function, SKP2B is expressed in founder cells, lateral root primordia and the root apical meristem. We identified a novel motif in the SKP2B promoter that is required for its specific root expression and auxin-dependent induction in the pericycle cells. Next to a transcriptional control by auxin, SKP2B expression is regulated by histone H3.1/H3.3 deposition in a CAF-dependent manner. The SKP2B promoter and the 5′ end of the transcribed region are enriched in H3.3, which is associated with active chromatin states, over H3.1. Furthermore, the SKP2B promoter is also regulated by H3 acetylation in an auxin- and IAA14-dependent manner, reinforcing the idea that epigenetics represents an important regulatory mechanism during lateral root formation.

The Emerging Role of Reactive Oxygen Species Signaling during Lateral Root Development

ROS signaling is crucial for lateral root emergence and root growth, and it regulates distinct sets of genes in these processes. Overall root architecture is the combined result of primary and lateral root growth and is influenced by both intrinsic genetic programs and external signals. One of the main questions for root biologists is how plants control the number of lateral root primordia and their emergence through the main root. We recently identified S-phase kinase-associated protein2 (SKP2B) as a new early marker for lateral root development. Here, we took advantage of its specific expression pattern in Arabidopsis (Arabidopsis thaliana) in a cell-sorting and transcriptomic approach to generate a lateral root-specific cell sorting SKP2B data set that represents the endogenous genetic developmental program. We first validated this data set by showing that many of the identified genes have a function during root growth or lateral root development. Importantly, genes encoding peroxidases were highly represented in our data set. Thus, we next focused on this class of enzymes and showed, using genetic and chemical inhibitor studies, that peroxidase activity and reactive oxygen species signaling are specifically required during lateral root emergence but, intriguingly, not for primordium specification itself.

Regulation of the New Arabidopsis Imprinted Gene AtBMI1C Requires the Interplay of Different Epigenetic Mechanisms

Recently, it has been shown that plants contain homologs to the animal Polycomb repressive complex 1 (PRC1) components BMI1 and RING1A/B. In Arabidopsis, there are three BMI1-like genes, two of which, AtBMI1A and B, are required during post-embryonic plant growth to repress embryonic traits and allow cell differentiation. However, little is known about the third BMI1-like gene, AtBMI1C. In this work, we show that AtBMI1C is only expressed during endosperm and stamen development. AtBMI1C is an imprinted gene expressed from the maternal allele in the endosperm but biallelically expressed in stamen. We found that the characteristic expression pattern of AtBMI1C is the result of a complex epigenetic regulation that involves CG DNA methylation, RNA-directed non-CG DNA methylation (RdDM), and PcG activity. Our results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

A Role for AUXIN RESISTANT3 in the Coordination of Leaf Growth

The characteristically flat structure of Arabidopsis thaliana vegetative leaves requires coordinating the growth of the epidermal, palisade mesophyll, spongy mesophyll and vascular tissues. Mutations disrupting such coordination or the specific growth properties of any of these tissues can cause hyponasty, epinasty, waviness or other deviations from flatness. Here, we show that the incurvata6 (icu6) semi-dominant allele of the AUXIN RESISTANT3 (AXR3) gene causes leaf hyponasty. Cotyledons and leaves of icu6/AXR3 plants exhibited reduced size of adaxial pavement cells, and abnormal expansion of palisade mesophyll cells. Enhanced auxin responses in the adaxial domain of icu6/AXR3 developing cotyledons and leaves correlated with increased cell divisions in the adaxial epidermis. Leaf incurvature in icu6/AXR3 leaves was alleviated by loss-of-function alleles of the ASYMMETRIC LEAVES1 (AS1) and AS2 genes, which restrict the expression of class I KNOX genes to the shoot apical meristem and regulate cell proliferation in leaf primordia. Taken together, our results suggest that an interaction between auxin responses and the AS1-AS2 pathway coordinates tissue growth during Arabidopsis thaliana leaf expansion.

Auxin and the ubiquitin pathway. Two players–one target: the cell cycle in action

Plants are sessile organisms that have to adapt their growth to the surrounding environment. Concomitant with this adaptation capability, they have adopted a post-embryonic development characterized by continuous growth and differentiation abilities. Constant growth is based on the potential of stem cells to divide almost incessantly and on a precise balance between cell division and cell differentiation. This balance is influenced by environmental conditions and by the genetic information of the cell. Among the internal cues, the cross-talk between different hormonal signalling pathways is essential to control this division/differentiation equilibrium. Auxin, one of the most important plant hormones, regulates cell division and differentiation, among many other processes. Amazing advances in auxin signal transduction at the molecular level have been reported, but how this signalling is connected to the cell cycle is, so far, not well known. Auxin signalling involves the auxin-dependent degradation of transcription repressors by F-box-containing E3 ligases of ubiquitin. Recently, SKP2A, another F-box protein, was shown to bind auxin and to target cell-cycle repressors for proteolysis, representing a novel mechanism that links auxin to cell division. In this review, a general vision of what is already known and the most recent advances on how auxin signalling connects to cell division and the role of the ubiquitin pathway in plant cell cycle will be covered.