Environmental control of rice flowering time.

Correct measurement of environmental parameters is fundamental for plant fitness and survival, as well as for timing developmental transitions, including the switch from vegetative to reproductive growth. Important parameters that affect flowering time include day length (photoperiod) and temperature. Their response pathways have been best described in Arabidopsis, which currently offers a detailed conceptual framework and serves as a comparison for other species. Rice, the focus of this review, also possesses a photoperiodic flowering pathway, but 150 million years of divergent evolution in very different environments have diversified its molecular architecture. The ambient temperature perception pathway is strongly intertwined with the photoperiod pathway and essentially converges on the same genes to modify flowering time. When observing network topologies, it is evident that the rice flowering network is centered on EARLY HEADING DATE 1, a rice-specific transcriptional regulator. Here, we summarize the most important features of the rice photoperiodic flowering network, with an emphasis on its uniqueness, and discuss its connections with hormonal, temperature perception, and stress pathways.

Molecular Links between Flowering and Abiotic Stress Response: A Focus on Poaceae.

Extreme temperatures, drought, salinity and soil pollution are the most common types of abiotic stresses crops can encounter in fields; these variations represent a general warning to plant productivity and survival, being more harmful when in combination. Plant response to such conditions involves the activation of several molecular mechanisms, starting from perception to signaling, transcriptional reprogramming and protein modifications. This can influence the plant's life cycle and development to different extents. Flowering developmental transition is very sensitive to environmental stresses, being critical to reproduction and to agricultural profitability for crops. The Poacee family contains some of the most widespread domesticated plants, such as wheat, barley and rice, which are commonly referred to as cereals and represent a primary food source. In cultivated Poaceae, stress-induced modifications of flowering time and development cause important yield losses by directly affecting seed production. At the molecular level, this reflects important changes in gene expression and protein activity. Here, we present a comprehensive overview on the latest research investigating the molecular pathways linking flowering control to osmotic and temperature extreme conditions in agronomically relevant monocotyledons. This aims to provide hints for biotechnological strategies that can ensure agricultural stability in ever-changing climatic conditions.

New Paradigms in Brassinosteroids, Strigolactones, Sphingolipids, and Nitric Oxide Interaction in the Control of Lateral and Adventitious Root Formation.

The root system is formed by the primary root (PR), which forms lateral roots (LRs) and, in some cases, adventitious roots (ARs), which in turn may produce their own LRs. The formation of ARs is also essential for vegetative propagation in planta and in vitro and for breeding programs. Root formation and branching is coordinated by a complex developmental network, which maximizes the plant's ability to cope with abiotic stress. Rooting is also a response caused in a cutting by wounding and disconnection from the donor plant. Brassinosteroids (BRs) are steroid molecules perceived at the cell surface. They act as plant-growth-regulators (PGRs) and modulate plant development to provide stress tolerance. BRs and auxins control the formation of LRs and ARs. The auxin/BR interaction involves other PGRs and compounds, such as nitric oxide (NO), strigolactones (SLs), and sphingolipids (SPLs). The roles of these interactions in root formation and plasticity are still to be discovered. SLs are carotenoid derived PGRs. SLs enhance/reduce LR/AR formation depending on species and culture conditions. These PGRs possibly crosstalk with BRs. SPLs form domains with sterols within cellular membranes. Both SLs and SPLs participate in plant development and stress responses. SPLs are determinant for auxin cell-trafficking, which is essential for the formation of LRs/ARs in planta and in in vitro systems. Although little is known about the transport, trafficking, and signaling of SPLs, they seem to interact with BRs and SLs in regulating root-system growth. Here, we review the literature on BRs as modulators of LR and AR formation, as well as their crosstalk with SLs and SPLs through NO signaling. Knowledge on the control of rooting by these non-classical PGRs can help in improving crop productivity and enhancing AR-response from cuttings.

Convergence between Development and Stress: Ectopic Xylem Formation in Arabidopsis Hypocotyl in Response to 24-Epibrassinolide and Cadmium.

Ectopic xylary element (EXE) formation in planta is a poorly investigated process, and it is unknown if it occurs as a response to the soil pollutant Cadmium (Cd). The pericycle cells of Arabidopsis thaliana hypocotyl give rise to EXEs under specific hormonal inputs. Cadmium triggers pericycle responses, but its role in EXE formation is unknown. Brassinosteroids (BRs) affect numerous developmental events, including xylogenesis in vitro, and their exogenous application by 24-epibrassinolide (eBL) helps to alleviate Cd-stress by increasing lateral/adventitious rooting. Epibrassinolide's effects on EXEs in planta are unknown, as well as its relationship with Cd in the control of the process. The research aims to establish an eBL role in pericycle EXE formation, a Cd role in the same process, and the possible interaction between the two. Results show that 1 nM eBL causes an identity reversal between the metaxylem and protoxylem within the stele, and its combination with Cd reduces the event. All eBL concentrations increase EXEs, also affecting xylary identity by changing from protoxylem to metaxylem in a concentration-dependent manner. Cadmium does not affect EXE identity but increases EXEs when combined with eBL. The results suggest that eBL produces EXEs to form a mechanical barrier against the pollutant.

Brassinosteroids Mitigate Cadmium Effects in Arabidopsis Root System without Any Cooperation with Nitric Oxide.

The heavy metal cadmium (Cd) affects root system development and quiescent center (QC)-definition in Arabidopsis root-apices. The brassinosteroids-(BRs)-mediated tolerance to heavy metals has been reported to occur by a modulation of nitric oxide (NO) and root auxin-localization. However, how BRs counteract Cd-action in different root types is unknown. This research aimed to find correlations between BRs and NO in response to Cd in Arabidopsis's root system, monitoring their effects on QC-definition and auxin localization in root-apices. To this aim, root system developmental changes induced by low levels of 24-epibrassinolide (eBL) or by the BR-biosynthesis inhibitor brassinazole (Brz), combined or not with CdSO4, and/or with the NO-donor nitroprusside (SNP), were investigated using morpho-anatomical and NO-epifluorescence analyses, and monitoring auxin-localization by the DR5::GUS system. Results show that eBL, alone or combined with Cd, enhances lateral (LR) and adventitious (AR) root formation and counteracts QC-disruption and auxin-delocalization caused by Cd in primary root/LR/AR apices. Exogenous NO enhances LR and AR formation in Cd-presence, without synergism with eBL. The NO-signal is positively affected by eBL, but not in Cd-presence, and BR-biosynthesis inhibition does not change the low NO-signal caused by Cd. Collectively, results show that BRs ameliorate Cd-effects on all root types acting independently from NO.

Responses to Cadmium in Early-Diverging Streptophytes (Charophytes and Bryophytes): Current Views and Potential Applications.

Several transition metals are essential for plant growth and development, as they are involved in various fundamental metabolic functions. By contrast, cadmium (Cd) is a metal that can prove extremely toxic for plants and other organisms in a dose-dependent manner. Charophytes and bryophytes are early-diverging streptophytes widely employed for biomonitoring purposes, as they are able to cope with high concentrations of toxic metal(loid)s without showing any apparent heavy damage. In this review, we will deal with different mechanisms that charophytes and bryophytes have evolved to respond to Cd at a cellular level. Particular attention will be addressed to strategies involving Cd vacuolar sequestration and cell wall immobilization, focusing on specific mechanisms that help achieve detoxification. Understanding the effects of metal(loid) pollution and accumulation on the morpho-physiological traits of charophytes and bryophytes can be in fact fundamental for optimizing their use as phytomonitors and/or phytoremediators.

Jasmonates, Ethylene and Brassinosteroids Control Adventitious and Lateral Rooting as Stress Avoidance Responses to Heavy Metals and Metalloids.

Developmental and environmental signaling networks often converge during plant growth in response to changing conditions. Stress-induced hormones, such as jasmonates (JAs), can influence growth by crosstalk with other signals like brassinosteroids (BRs) and ethylene (ET). Nevertheless, it is unclear how avoidance of an abiotic stress triggers local changes in development as a response. It is known that stress hormones like JAs/ET and BRs can regulate the division rate of cells from the first asymmetric cell divisions (ACDs) in meristems, suggesting that stem cell activation may take part in developmental changes as a stress-avoidance-induced response. The root system is a prime responder to stress conditions in soil. Together with the primary root and lateral roots (LRs), adventitious roots (ARs) are necessary for survival in numerous plant species. AR and LR formation is affected by soil pollution, causing substantial root architecture changes by either depressing or enhancing rooting as a stress avoidance/survival response. Here, a detailed overview of the crosstalk between JAs, ET, BRs, and the stress mediator nitric oxide (NO) in auxin-induced AR and LR formation, with/without cadmium and arsenic, is presented. Interactions essential in achieving a balance between growth and adaptation to Cd and As soil pollution to ensure survival are reviewed here in the model species Arabidopsis and rice.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice.

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor-/- knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor-/-/Dnmt3a-/- knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family.

Potato virus X (PVX) is a positive-sense single-stranded RNA (ssRNA) filamentous plant virus belonging to the Alphaflexiviridae family, considered in recent years as a tool for nanotechnology applications. We present the cryo-electron microscopy structure of the PVX particle at a resolution of 2.2 Å. The well-defined density of the coat proteins and of the genomic RNA allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules. The particle is formed by repeated segments made of 8.8 coat proteins, forming a left-handed helical structure. The RNA runs in an internal crevice along the virion, packaged in 5-nucleotide repeats in which the first four bases are stacked in the classical way, while the fifth is rotated and nearly perpendicular. The resolution of the structure described here suggests a mechanism for the virion assembly and potentially provides a platform for the rational design of antiviral compounds and for the use of PVX in nanotechnology.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Jasmonic Acid Methyl Ester Induces Xylogenesis and Modulates Auxin-Induced Xylary Cell Identity with NO Involvement.

In Arabidopsis basal hypocotyls of dark-grown seedlings, xylary cells may form from the pericycle as an alternative to adventitious roots. Several hormones may induce xylogenesis, as Jasmonic acid (JA), as well as indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) auxins, which also affect xylary identity. Studies with the ethylene (ET)-perception mutant ein3eil1 and the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC), also demonstrate ET involvement in IBA-induced ectopic metaxylem. Moreover, nitric oxide (NO), produced after IBA/IAA-treatments, may affect JA signalling and interact positively/negatively with ET. To date, NO-involvement in ET/JA-mediated xylogenesis has never been investigated. To study this, and unravel JA-effects on xylary identity, xylogenesis was investigated in hypocotyls of seedlings treated with JA methyl-ester (JAMe) with/without ACC, IBA, IAA. Wild-type (wt) and ein3eil1 responses to hormonal treatments were compared, and the NO signal was quantified and its role evaluated by using NO-donors/scavengers. Ectopic-protoxylem increased in the wt only after treatment with JAMe(10 µM), whereas in ein3eil1 with any JAMe concentration. NO was detected in cells leading to either xylogenesis or adventitious rooting, and increased after treatment with JAMe(10 µM) combined or not with IBA(10 µM). Xylary identity changed when JAMe was applied with each auxin. Altogether, the results show that xylogenesis is induced by JA and NO positively regulates this process. In addition, NO also negatively interacts with ET-signalling and modulates auxin-induced xylary identity.

Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS.

Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 µl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8-91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Selective Knockdowns in Maize by Sequence-Specific Protein Aggregation.

Protein aggregation is determined by 5-15 amino acids peptides of the target protein sequence, so-called aggregation-prone regions (APRs) that specifically self-associate to form ß-structured inclusions. The presence of APRs in a target protein can be predicted by a dedicated algorithm, such as TANGO. Synthetic aggregation-prone proteins are designed by expressing specific APRs fused to a fluorescent carrier for stability and visualization. Previously, the stable expression of these proteins in Zea mays (maize) has been demonstrated to induce aggregation of target proteins with specific localization, such as the starch-degrading enzyme α-glucan water dikinase, giving rise to plants displaying knockdown phenotypes. Here, we describe how to design synthetic aggregation-prone proteins to harness the sequence specificity of APRs to generate aggregation-associated phenotypes in a targeted manner and in different subcellular compartments. This method points toward the application of induced targeted aggregation as a useful tool to knock down protein functions in maize and to generate crops with improved traits.

Sequence-Specific Protein Aggregation Generates Defined Protein Knockdowns in Plants.

Protein aggregation is determined by short (5-15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form ß-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme α-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops.

Brassinosteroid production and signaling differentially control cell division and expansion in the leaf.

Brassinosteroid (BR) hormones control plant growth through acting on both cell expansion and division. Here, we examined the role of BRs in leaf growth using the Arabidopsis BR-deficient mutant constitutive photomorphogenesis and dwarfism (cpd). We show that the reduced size of cpd leaf blades is a result of a decrease in cell size and number, as well as in venation length and complexity. Kinematic growth analysis and tissue-specific marker gene expression revealed that the leaf phenotype of cpd is associated with a prolonged cell division phase and delayed differentiation. cpd-leaf-rescue experiments and leaf growth analysis of BR biosynthesis and signaling gain-of-function mutants showed that BR production and BR receptor-dependent signaling differentially control the balance between cell division and expansion in the leaf. Investigation of cell cycle markers in leaves of cpd revealed the accumulation of mitotic proteins independent of transcription. This correlated with an increase in cyclin-dependent kinase activity, suggesting a role for BRs in control of mitosis.

Brassinosteroids tailor stomatal production to different environments.

Two recent reports show that brassinosteroids control stomata production by regulating the GSK3-like kinase BIN2-mediated phosphorylation of two different stomatal signalling components resulting in opposite stomatal phenotypes. We discuss how these two mechanisms might differentially control stomatal generation under diverse growth conditions.

SPEECHLESS integrates brassinosteroid and stomata signalling pathways.

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.