Concepción Manzano

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.

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.

Flavonols Mediate Root Phototropism and Growth through Regulation of Proliferation-to-Differentiation Transition

Light locally induces the accumulation of flavonols to promote cell elongation and asymmetric growth in the root transition zone, suggesting that flavonols serve as positional signals. Roots normally grow in darkness, but they may be exposed to light. After perceiving light, roots bend to escape from light (root light avoidance) and reduce their growth. How root light avoidance responses are regulated is not well understood. Here, we show that illumination induces the accumulation of flavonols in Arabidopsis thaliana roots. During root illumination, flavonols rapidly accumulate at the side closer to light in the transition zone. This accumulation promotes asymmetrical cell elongation and causes differential growth between the two sides, leading to root bending. Furthermore, roots illuminated for a long period of time accumulate high levels of flavonols. This high flavonol content decreases both auxin signaling and PLETHORA gradient as well as superoxide radical content, resulting in reduction of cell proliferation. In addition, cytokinin and hydrogen peroxide, which promote root differentiation, induce flavonol accumulation in the root transition zone. As an outcome of prolonged light exposure and flavonol accumulation, root growth is reduced and a different root developmental zonation is established. Finally, we observed that these differentiation-related pathways are required for root light avoidance. We propose that flavonols function as positional signals, integrating hormonal and reactive oxygen species pathways to regulate root growth direction and rate in response to light.

D-Root: a system for cultivating plants with the roots in darkness or under different light conditions

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.

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.

Post-embryonic organogenesis and plant regeneration from tissues: two sides of the same coin?

Plants have extraordinary developmental plasticity as they continuously form organs during post-embryonic development. In addition they may regenerate organs upon in vitro hormonal induction. Advances in the field of plant regeneration show that the first steps of de novo organogenesis through in vitro culture in hormone containing media (via formation of a proliferating mass of cells or callus) require root post-embryonic developmental programs as well as regulators of auxin and cytokinin signaling pathways. We review how hormonal regulation is delivered during lateral root initiation and callus formation. Implications in reprograming, cell fate and pluripotency acquisition are discussed. Finally, we analyze the function of cell cycle regulators and connections with epigenetic regulation. Future work dissecting plant organogenesis driven by both endogenous and exogenous cues (upon hormonal induction) may reveal new paradigms of common regulation.

SKP2A protein, an F-box that regulates cell division, is degraded via the ubiquitin pathway

The ubiquitin pathway is emerging as a powerful system that controls the stability of key regulatory proteins. In plants, this pathway plays an important role in controlling several developmental processes, responses to environmental changes and also cell division. Arabidopsis SKP2A is an F-box protein that regulates the stability of the E2FC-DPB transcription factor, a repressor of cell proliferation. Although the function of SKP2A is to recruit targets for degradation, we have shown that SKP2A is also degraded through the Ub/26S pathway and, interestingly, auxin stimulates such degradation. Overexpression of SKP2A positively regulates cell division, increasing the number of cells in G2/M, reducing the level of ploidy and developing higher number of lateral root primordia. In addition, we showed in this report that overexpression of SKP2A increased the survival of Arabidopsis plants when they grown on a medium with high levels of sucrose, likely by maintaining cell division active. Thus, it is likely that SKP2A connects cell division with stress responses. Addendum to: Jurado S, Díaz-Triviño S, Abraham Z, Manzano C, Gutierrez C, Del Pozo C. SKP2A, an F-box protein that regulates cell division, is degraded via the ubiquitin pathway. Plant J 2008; 53:828-41.

Control of Arabidopsis lateral root primordium boundaries by MYB36

Root branching in plants relies on the de novo formation of lateral roots. These are initiated from founder cells, triggering new formative divisions that generate lateral root primordia (LRP). The LRP size and shape depends on the balance between positive and negative signals that control cell proliferation. The mechanisms controlling proliferation potential of LRP cells remains poorly understood. We found that Arabidopsis thaliana MYB36, which have been previously shown to regulate genes required for Casparian strip formation and the transition from proliferation to differentiation in the primary root, plays a new role in controlling LRP development at later stages. We found that MYB36 is a novel component of LR development at later stages. MYB36 was expressed in the cells surrounding LRP where it controls a set of peroxidase genes, which maintain reactive oxygen species (ROS) balance. This was required to define the transition between proliferating and arrested cells inside the LRP, coinciding with the change from flat to dome-shaped primordia. Reducing the levels of hydrogen peroxide (H2 O2 ) in myb36-5 significantly rescues the mutant phenotype. Our results uncover a role for MYB36 outside the endodermis during LRP development through a mechanism analogous to regulating the proliferation/differentiation transition in the root meristem.

A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development

Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.