Role of Arabidopsis RabG3b and autophagy in tracheary element differentiation

The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid-insensitive mutant bri1-301 and an autophagy mutant atg5-1. On the basis of our results, we propose that RabG3b functions as a component of autophagy and regulates TE differentiation by activating the process of PCD.     

Programmed cell death of tracheary elements as a paradigm in plants

Plant development involves various programmed cell death (PCD) processes. Among them, cell death occurring during differentiation of procambium into tracheary elements (TEs), which are a major component of vessels or tracheids, has been studied extensively. Recent studies of PCD during TE differentiation mainly using an in vitro differentiation system of Zinnia have revealed that PCD of TEs is a plant-specific one in which the vacuole plays a central role. Furthermore, there are recent findings of several factors that may initiate PCD of TEs and that act at autonomous degradation of cell contents. Herein I summarize the present knowledge about cell death program during TE differentiation as an excellent example of PCD in plants.     

Role of an <Emphasis Type="Italic">Arabidopsis</Emphasis> Rab GTPase RabG3b in Pathogen Response and Leaf Senescence

In our previous proteomic analysis, we isolated a small GTPase RabG3b as a salicylic acid-responsive protein in Arabidopsis (Oh et al. in Plant Cell 17:2832–2847, 2005). Here, we constructed transgenic plants overexpressing wild-type (RabG3bOX), constitutively active (RabG3bCA), and dominant negative (RabG3bDN) forms of RabG3b for functional studies. The phenotypes of these transgenic plants were indistinguishable from wild-type plants under normal growth conditions. However, both RabG3bOX and RabG3bCA plants displayed unrestricted hypersensitive programmed cell death against a fungal toxin Fumonisin B1 and a fungal pathogen Alternaria brassicicola, whereas no major difference between wild-type and RabG3bDN plants was observed. In addition, RabG3bOX and RabG3bCA plants underwent accelerated leaf senescence compared to wild-type and RabG3bDN plants. These results suggest that RabG3b is a modulator for cell death progression during pathogen response and senescence process in plants. An erratum to this article can be found at     

Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba × Populus tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed a range of morphological, histochemical and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild-type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD- and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.     

Tracheary element differentiation in Zinnia mesophyll cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans differentiate into tracheary elements (TEs) when cultured in a medium containing adequate auxin and cytokinin. Differentiation in this culture system is relatively synchronous, rapid (occuring within 3 days of cell isolation) and efficient (with up to 65% of the mesophyll cells differentiating into TEs), and does not require prior mitosis. The Zinnia system has been used to investigate (a) cytological and ultrastructural changes occurring during TE differentiation, such as the reorganization of microtubules controlling secondary wall deposition, (b) the influences of calcium and of various plant hormones and antihormones on TE differentiation, and (c) biochemical changes during differentiation, including those occurring during secondary wall deposition, lignification and autolysis. This review summarizes experiments in which the Zinnia system has served as a model for the study of TE differentiation.     

Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (<Emphasis Type="Italic">Zinnia elegans</Emphasis>) cell cultures

Background  

The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented.     

Age‐associated de‐repression of retrotransposons in the Drosophila fat body,its potential cause and consequence

Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin‐mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age‐associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de‐repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin‐B in the old fat body cells. Depletion of the Drosophila lamin‐B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin‐B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons.     

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10⁵ cells ml⁻¹. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25–300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated “xylogen” with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12–36 h before visible thickening of secondary cell walls of TEs). Received: 13 July 2000 / Accepted: 4 October 2000     

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions

Background and Aims

Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings.

Methods

Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H₂O₂) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ.

Key Results

The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H₂O₂ synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole.

Conclusions

The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.     

Loss of Tonoplast Integrity Programmed in Tracheary Element Differentiation

A tracheary element (TE) is a typical example of a cell type that undergoes programmed cell death in the developmental processes of vascular plants. The loss of the selective permeability of the tonoplast, which corresponds to tonoplast disintegration, occurred after the cells commenced secondary wall thickening and played a pivotal role in the programmed cell death of TEs in a zinnia (Zinnia elegans L.) cell culture. A search for events specifically associated with the TE vacuole provided an important clue to the understanding of the cell death mechanism. The transport of fluorescein, a fluorescent organic anion, across the tonoplast declined drastically in differentiating TEs. The capacity of the vacuole to accumulate the probe was also impaired. Treatment with probenecid, an inhibitor of organic anion transport, caused rapid cell death of TEs and led to the ultimate disruption of the vacuole even in other types of cultured cells. These changes in vacuolar properties during TE development were suppressed by cycloheximide. Specific mRNA accumulation in cells cultured in a TE differentiation-inductive condition was abolished by probenecid. These results suggest that a change in vacuolar membrane permeability promotes programmed cell death in TEs.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Regulation of SOBIR1 accumulation and activation of defense responses in bir1–1 by specific components of ER quality control

Receptor‐like kinases play diverse roles in plant biology. Arabidopsis BAK1‐INTERACTING RECEPTOR‐LIKE KINASE 1 (BIR1) functions as a negative regulator of plant immunity. bir1‐1 mutant plants display spontaneous cell death and constitutive defense responses that are dependent on SUPPRESSOR OF BIR1,1 (SOBIR1) and PHYTOALEXIN DEFICIENT4 (PAD4). Here we report that mutations in three components of ER quality control, CALRETICULIN3 (CRT3), ER‐LOCALIZED DnaJ‐LIKE PROTEIN 3b (ERdj3b) and STROMAL‐DERIVED FACTOR‐2 (SDF2), also suppress the spontaneous cell death and constitutive defense responses in bir1‐1. Further analysis revealed that accumulation of the SOBIR1 protein is reduced in crt3‐1 and erdj3b‐1 mutant plants. These data suggest that ER quality control plays important roles in the biogenesis of SOBIR1, and is required for cell death and defense responses in bir1‐1.     

Transposon variation by order during allopolyploidisation between Brassica oleracea and Brassica rapa

Although many studies have shown that transposable element (TE) activation is induced by hybridisation and polyploidisation in plants, much less is known on how different types of TE respond to hybridisation, and the impact of TE‐associated sequences on gene function. We investigated the frequency and regularity of putative transposon activation for different types of TE, and determined the impact of TE‐associated sequence variation on the genome during allopolyploidisation. We designed different types of TE primers and adopted the Inter‐Retrotransposon Amplified Polymorphism (IRAP) method to detect variation in TE‐associated sequences during the process of allopolyploidisation between Brassica rapa (AA) and Brassica oleracea (CC), and in successive generations of self‐pollinated progeny. In addition, fragments with TE insertions were used to perform Blast2GO analysis to characterise the putative functions of the fragments with TE insertions. Ninety‐two primers amplifying 548 loci were used to detect variation in sequences associated with four different orders of TE sequences. TEs could be classed in ascending frequency into LTR‐REs, TIRs, LINEs, SINEs and unknown TEs. The frequency of novel variation (putative activation) detected for the four orders of TEs was highest from the F₁ to F₂ generations, and lowest from the F₂ to F₃ generations. Functional annotation of sequences with TE insertions showed that genes with TE insertions were mainly involved in metabolic processes and binding, and preferentially functioned in organelles. TE variation in our study severely disturbed the genetic compositions of the different generations, resulting in inconsistencies in genetic clustering. Different types of TE showed different patterns of variation during the process of allopolyploidisation.     

Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

Mechanisms for shaping,orienting, positioning and patterning plant secondary cell walls

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that—as for the growth of all plant organs—are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls.Key words: xylem/wood vessels, tracheary elements, secondary cell wall, cell wall patterning, microtubules, microtubule-associated proteins, MAP70Xylem formation has been one of the key steps of plant evolution. These physically strong tube cells allowed plants to colonize land by reinforcing their upright position against gravity and resisting desiccation by permitting water conduction throughout the plant body. This double role is fulfilled by specific conducting wood cells—the tracheary elements (TEs). These cells represent the cellular units of the adjustable plant vasculature, which relies on the three structural characteristics of TEs: (1) these cells develop a secondary cell wall to resist pressure exerted by the sap they will conducted, (2) these cells undergo programmed cell death (PCD) to hollow out their entire cytoplasmic content to form a conduit for the sap and (3) these cells will undergo a terminal perforation at their basal end (with respect to the corresponding meristem) to form a complete functional vascular cylinder which will connect with the underlying vascular vessels once terminally differentiated.^1,2 TEs are further characterized by a diversity of organizational pattern described by their secondary cell wall, which can be annular or spiral (referred to as protoxylem-type ornamentations) reticulate or pitted (referred to as metaxylem-type ornamentations).^3,4 These differently ornamented TEs are developmentally regulated and for protoxylemtype TEs appear during the development of early primary tissues (annular TEs are mostly observed in developing embryos) while metaxylem-type TEs appear in the later development of primary and secondary tissues (they represent the TEs present in wood). Annular and spiral TEs are first formed in organs undergoing primary growth and are considered to be “extendable” (their pattern in rings and spirals does not oppose further extension of the TE cell) during the growth of this organ. Once the growing organ has attained a certain size these TEs will be crushed by the surrounding tissue whilst the more heavily reinforced reticulate and pitted TEs will form to insure the vascular flow and strengthen the entire organ. In short, the modularity and plasticity of this plant vascular system is directly dependant on the differentiation and the type of cell wall ornamentation of its constituent TEs. The establishment of such regular patterning of secondary cell walls has been attributed to the underlying cortical microtubule array that predefines the cell wall depositions (reviewed in ref. ²). Pharmacological modulation of microtubule properties in both whole plants and in vitro TE differentiating systems leads to severe defects in the patterning, orientation, smoothness and deposition of TE secondary cell walls (reviewed in ref. ²).     

Involvement of NtERF3 in the cell death signalling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants

We previously reported that one of the ethylene response factors (ERFs), NtERF3, and other members of the subgroup VIII‐a ERFs of the AP2/ERF family exhibit cell death‐inducing ability in tobacco leaves. In this study, we focused on the involvement of NtERF3 in a cell death signalling pathway in tobacco plants, particularly downstream of NtSIPK/NtWIPK and NtWRKY1, which are mitogen‐activated protein kinases and a phosphorylation substrate of NtSIPK, respectively. An ERF‐associated amphiphilic repression (EAR) motif‐deficient NtERF3b mutant (NtERF3bΔEAR) that lacked cell death‐inducing ability suppressed the induction of cell death caused by NtERF3a. The transient co‐expression of NtERF3bΔEAR suppressed the hypersensitive reaction (HR)‐like cell death induced by NtSIPK and NtWRKY1. The induction of cell death by NtSIPK and NtWRKY1 was also inhibited in transgenic plants expressing NtERF3bΔEAR. Analysis of gene expression, ethylene production and cell death symptoms in salicylic acid‐deficient tobacco plants suggested the existence of some feedback regulation in the HR cell death signalling pathway mediated by SIPK/WIPK and WRKY1. Overall, these results suggest that NtERF3 functions downstream of NtSIPK/NtWIPK and NtWRKY1 in a cell death signalling pathway, with some feedback regulation.