Genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with herbicide resistance evolved before the origin of land plants.

Cytochrome P450 (CYP) monooxygenases and glutathione S-transferases (GST) are enzymes that catalyse chemical modifications of a range of organic compounds. Herbicide resistance has been associated with higher levels of CYP and GST gene expression in some herbicide-resistant weed populations compared to sensitive populations of the same species. By comparing the protein sequences of 9 representative species of the Archaeplastida-the lineage which includes red algae, glaucophyte algae, chlorophyte algae, and streptophytes-and generating phylogenetic trees, we identified the CYP and GST proteins that existed in the common ancestor of the Archaeplastida. All CYP clans and all but one land plant GST classes present in land plants evolved before the divergence of streptophyte algae and land plants from their last common ancestor. We also demonstrate that there are more genes encoding CYP and GST proteins in land plants than in algae. The larger numbers of genes among land plants largely results from gene duplications in CYP clans 71, 72, and 85 and in the GST phi and tau classes [1,2]. Enzymes that either metabolise herbicides or confer herbicide resistance belong to CYP clans 71 and 72 and the GST phi and tau classes. Most CYP proteins that have been shown to confer herbicide resistance are members of the CYP81 family from clan 71. These results demonstrate that the clan and class diversity in extant plant CYP and GST proteins had evolved before the divergence of land plants and streptophyte algae from a last common ancestor estimated to be between 515 and 474 million years ago. Then, early in embryophyte evolution during the Palaeozoic, gene duplication in four of the twelve CYP clans, and in two of the fourteen GST classes, led to the large numbers of CYP and GST proteins found in extant land plants. It is among the genes of CYP clans 71 and 72 and GST classes phi and tau that alleles conferring herbicide resistance evolved in the last fifty years.

Fifteen compelling open questions in plant cell biology.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants.

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.

PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis.

Root hairs elongate by tip growth and simultaneously harden the shank by constructing the inner secondary cell wall layer. While much is known about the process of tip growth1, almost nothing is known about the mechanism by which root hairs harden the shank. Here we show that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), the enzymatic product of FORMATION OF APLOID AND BINUCLEATE CELLS 1 (FAB1), is involved in the hardening of the shank in root hairs in Arabidopsis. FAB1 and PtdIns(3,5)P2 localize to the plasma membrane along the shank of growing root hairs. By contrast, phosphatidylinositol 4-phosphate 5-kinase 3 (PIP5K3) and PtdIns(4,5)P2 localize to the apex of the root hair where they are required for tip growth. Reduction of FAB1 function results in the formation of wavy root hairs while those of the wild type are straight. The localization of FAB1 in the plasma membrane of the root hair shank requires the activity of Rho-related GTPases from plants 10 (ROP10) and localization of ROP10 requires FAB1 activity. Computational modelling of root hair morphogenesis successfully reproduces the wavy root hair phenotype. Taken together, these data demonstrate that root hair shank hardening requires PtdIns(3,5)P2/ROP10 signalling.

Growth regulation in tip-growing cells that develop on the epidermis.

Plants develop tip-growing extensions-root hairs and rhizoids-that initiate as swellings on the outer surface of individual epidermal cells. A conserved genetic mechanism controls the earliest stages in the initiation of these swellings. The same mechanism controls the formation of multicellular structures that develop from swellings on epidermal cells in early diverging land plants. Details of the molecular events that regulate the positioning of the swellings involve sterols and phosphatidylinositol phosphates. The final length of root hairs is determined by the intensity of a pulse of transcription factor synthesis. Genes encoding similar transcription factors control root hair development in cereals and are potential targets for crop improvement.

A mutual support mechanism through intercellular movement of CAPRICE and GLABRA3 can pattern the Arabidopsis root epidermis.

The patterning of the Arabidopsis root epidermis depends on a genetic regulatory network that operates both within and between cells. Genetic studies have identified a number of key components of this network, but a clear picture of the functional logic of the network is lacking. Here, we integrate existing genetic and biochemical data in a mathematical model that allows us to explore both the sufficiency of known network interactions and the extent to which additional assumptions about the model can account for wild-type and mutant data. Our model shows that an existing hypothesis concerning the autoregulation of WEREWOLF does not account fully for the expression patterns of components of the network. We confirm the lack of WEREWOLF autoregulation experimentally in transgenic plants. Rather, our modelling suggests that patterning depends on the movement of the CAPRICE and GLABRA3 transcriptional regulators between epidermal cells. Our combined modelling and experimental studies show that WEREWOLF autoregulation does not contribute to the initial patterning of epidermal cell fates in the Arabidopsis seedling root. In contrast to a patterning mechanism relying on local activation, we propose a mechanism based on lateral inhibition with feedback. The active intercellular movements of proteins that are central to our model underlie a mechanism for pattern formation in planar groups of cells that is centred on the mutual support of two cell fates rather than on local activation and lateral inhibition.

Ethylene modulates stem cell division in the Arabidopsis thaliana root.

The construction of multicellular organisms depends on stem cells-cells that can both regenerate and produce daughter cells that undergo differentiation. Here, we show that the gaseous messenger ethylene modulates cell division in the cells of the quiescent center, which act as a source of stem cells in the seedling root. The cells formed through these ethylene-induced divisions express quiescent center-specific genes and can repress differentiation of surrounding initial cells, showing that quiescence is not required for these cells to signal to adjacent stem cells. We propose that ethylene is part of a signaling pathway that modulates cell division in the quiescent center in the stem cell niche during the postembryonic development of the root system.

Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens.

Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.

Three-dimensional modelling of wheat endosperm development.

We have developed methods, based on confocal microscopy and three-dimensional (3D) modelling, for the analysis of complex tissues and individual nuclei. These methods were used to study the development of early wheat (Triticum aestivum) endosperm as a whole and of endosperm nuclei undergoing polyploidization. Fixed sections of immature caryopses were either stained with SYTOX Green or used for fluorescence in situ hybridization (FISH) to visualize centromeres, telomeres and a rye chromosome arm substitution. Each section was imaged as a confocal image stack. By using Amira 3.0 for computer image processing, rendered models were produced of the whole endosperm and of individual nuclei. We followed endosperm development up to the formation of a complete syncytium, which develops via a dorsal and a ventral plate of nuclei in the central cell. Modelling of nuclei showed that wheat chromosomes are not anchored to the nuclear membrane and become more randomly positioned in endoreduplicated nuclei. This analysis produced a precise description of the positioning of nuclei throughout the developing endosperm and of chromosomal domains in single nuclei.

Epidermal patterning genes are active during embryogenesis in Arabidopsis.

Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3--a katanin-p60 protein.

The Arabidopsis root is composed of radial cell layers, each with distinct identities. The epidermal layer is composed of rows of hair cells flanked on either side by rows of non-hair epidermal cells. The development of hair and non-hair cells is dependent on domains of positional information with strict boundaries. The pattern of cell differentiation and the expression of molecular markers of cell fate is altered in the ectopic root hair 3 (erh3) mutant epidermis indicating that ERH3 is required for the specification of cell fates from early in development (in the meristem) through differentiation. Furthermore the expression of molecular markers indicates that the specification of cell identities is defective within other radial cell layers. ERH3 encodes a p60 katanin protein that is expressed throughout the plant. Katanin proteins are known to sever microtubules, and have a role in the organisation of the plant cell wall since mutants with decreased katanin activity have been shown to have defective walls. We suggest that microtubules are involved in the specification of cell identities in cells of the Arabidopsis root. Microtubules may be required for the localization of positional cues in the wall that have previously been shown to operate in the development of the root epidermis. Alternatively microtubules may be involved in another as yet undefined process required for the specification of cell identity in plants.