Federica Della Rovere

Oligogalacturonides enhance cytokinin-induced vegetative shoot formation in tobacco explants, inhibit polyamine biosynthetic gene expression, and promote long-term remobilisation of cell calcium

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca2+-dependent “egg-box” conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl2 in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca2+ and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification

AbstractMain conclusionThe heterologous expression ofAtPCS1in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

The Arabidopsis BET bromodomain factor GTE4 is involved in maintenance of the mitotic cell cycle during plant development

Bromodomain and Extra Terminal domain (BET) proteins are characterized by the presence of two types of domains, the bromodomain and the extra terminal domain. They bind to acetylated lysines present on histone tails and control gene transcription. They are also well known to play an important role in cell cycle regulation. In Arabidopsis (Arabidopsis thaliana), there are 12 BET genes; however, only two of them, IMBIBITION INDUCIBLE1 and GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6), were functionally analyzed. We characterized GTE4 and show that gte4 mutant plants have some characteristic features of cell cycle mutants. Their size is reduced, and they have jagged leaves and a reduced number of cells in most organs. Moreover, cell size is considerably increased in the root, and, interestingly, the root quiescent center identity seems to be partially lost. Cell cycle analyses revealed that there is a delay in activation of the cell cycle during germination and a premature arrest of cell proliferation, with a switch from mitosis to endocycling, leading to a statistically significant increase in ploidy levels in the differentiated organs of gte4 plants. Our results point to a role of GTE4 in cell cycle regulation and specifically in the maintenance of the mitotic cell cycle.

The quiescent center and the stem cell niche in the adventitious roots of Arabidopsis thaliana.

ABSTRACT Adventitious rooting is essential for the survival of numerous species from vascular cryptogams to monocots, and is required for successful micropropagation. The tissues involved in AR initiation may differ in planta and in in vitro systems. For example, in Arabidopsis thaliana, ARs originate from the hypocotyl pericycle in planta and the stem endodermis in in vitro cultured thin cell layers. The formation of adventitious roots (ARs) depends on numerous factors, among which the hormones, auxin, in particular. In both primary and lateral roots, growth depends on a functional stem cell niche in the apex, maintained by an active quiescent center (QC), and involving the expression of genes controlled by auxin and cytokinin. This review summarizes current knowledge about auxin and cytokinin control on genes involved in the definition and maintenance of QC, and stem cell niche, in the apex of Arabidopsis ARs in planta and in longitudinal thin cell layers.

Recent Advances on Genetic and Physiological Bases of In Vitro Somatic Embryo Formation.

Somatic embryogenesis involves a broad repertoire of genes, and complex expression patterns controlled by a concerted gene regulatory network. The present work describes this regulatory network focusing on the main aspects involved, with the aim of providing a deeper insight into understanding the total reprogramming of cells into a new organism through a somatic way. To the aim, the chromatin remodeling necessary to totipotent stem cell establishment is described, as the activity of numerous transcription factors necessary to cellular totipotency reprogramming. The eliciting effects of various plant growth regulators on the induction of somatic embryogenesis is also described and put in relation with the activity of specific transcription factors. The role of programmed cell death in the process, and the related function of specific hemoglobins as anti-stress and anti-death compounds is also described. The tools for biotechnology coming from this information is highlighted in the concluding remarks.

Plant architecture, auxin homeostasis and phenol content in Arabidopsis thaliana grown in cadmium- and zinc-enriched media

A screening strategy using micropropagation glass tubes with a gradient of distances between germinating seeds and a metal-contaminated medium was used for studying alterations in root architecture and morphology of Arabidopsis thaliana treated with cadmium (Cd) and zinc (Zn) at the concentration of 10-20μM and 100-200μM, respectively. Metal concentrations in plant shoots and roots were measured by quadrupole inductively coupled plasma mass spectrometry. After 21days from germination, all plants in the tubes were scanned at high resolution and the root systems analyzed. The localization of indole-3-acetic acid (IAA) in the primary root and lateral root apices was monitored using DR5:GUS, LAX3:GUS and AUX1:GUS Arabidopsis transgenic lines. Total phenol content in leaves was measured spectrophotometrically. Shoot and root dry weight and leaf area did not change in Zn-exposed plants and significantly decreased in Cd-exposed plants, compared to control plants. Cadmium induced a reduction of root length, of mean number of roots and of total root surface. Both Cd- and Zn-exposed plants showed a reduced specific root length. This morphological behavior, together with an observed increase in root diameter in metal-exposed plants could be interpreted as compensatory growth, and the observed thicker roots could act as a barrier to protect root from the metals. In comparison with the apical localization of the IAA signal in the control plants, Zn generally reinforced the intensity of IAA signal, without affecting its localization. In Cd-exposed plants, IAA localization remained apical but weaker compared to control plants. Total phenols decreased in plants exposed to Zn and Cd. Therefore, we propose that the remodelling of the root architecture and the production of some secondary metabolites, such as IAA and phenols could be two responses of plants subjected to metal stress. This knowledge can open the way to future phytoremediation strategies of contaminated sites.

Indole-3-Butyric Acid Induces Ectopic Formation of Metaxylem in the Hypocotyl of Arabidopsis thaliana without Conversion into Indole-3-Acetic Acid and with a Positive Interaction with Ethylene

The role of the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) and of the auxin-interacting phytohormone ethylene, on the ectopic formation of primary xylem (xylogenesis in planta) is still little known. In particular, auxin/ethylene-target tissue(s), modality of the xylary process (trans-differentiation vs. de novo formation), and the kind of ectopic elements formed (metaxylem vs. protoxylem) are currently unknown. It is also unclear whether IBA may act on the process independently of conversion into IAA. To investigate these topics, histological analyses were carried out in the hypocotyls of Arabidopsis wild type seedlings and ech2ibr10 and ein3eil1 mutants, which are blocked in IBA-to-IAA conversion and ethylene signalling, respectively. The seedlings were grown under darkness with either IAA or IBA, combined or not with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Adventitious root formation was also investigated because this process may compete with xylogenesis. Our results show that ectopic formation of protoxylem and metaxylem occurred as an indirect process starting from the pericycle periclinal derivatives of the hypocotyl basal part. IAA favoured protoxylem formation, whereas IBA induced ectopic metaxylem with ethylene cooperation through the EIN3EIL1 network. Ectopic metaxylem differentiation occurred independently of IBA-to-IAA conversion as mediated by ECH2 and IBR10, and in the place of IBA-induced adventitious root formation.

The Arabidopsis BET bromodomain factor GTE4 regulates the mitotic cell cycle

Proteins containing bromodomains are capable of binding to acetylated histone tails and have a role in recognizing and deciphering acetylated chromatin. Plant BET proteins contain one bromodomain. Twelve BET-encoding genes have been identified in the Arabidopsis genome. Two of these genes have been functionally characterized, one shows a role in seed germination, the other is involved in the establishment of leaf shape. Recently, we characterized a third AtBET gene, named GTE4. We demonstrated that GTE4 is involved in the activation and maintenance of cell division in the meristems and by this controls cell numbers in differentiated organs. Moreover, the quiescent center (QC) identity is partially lost in the apex of the primary root of gte4 mutant, and there is a premature switch from mitosis to endocycling. Genes involved in the retinoblastoma (RB)-E2F pathway, which is important for coupling cell division and cell differentiation in plants and animals, were either up- or down-regulated in the gte4 mutant. In this report we also show that the defect in germination observed in gte4 mutant seeds is not rescued by the action of GA3. Further the root pole of the mutant embryo shows irregular cytokinesis in the procambial stem cells, and the QC of the lateral root shows a partial, but not transient, loss of QC identity. These additional results reinforce the importance of GTE4 in the control of cell proliferation.