Control of lateral root formation by rapamycin-induced dimerization of engineered RGI/BAK1 and by BIR3 chimera.

Leucine-rich repeat receptor kinases (LRR-RKs) play a pivotal role in diverse aspects of growth, development, and immunity in plants by sensing extracellular signals. Typically, LRR-RKs are activated through the ligand-induced interaction with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) coreceptor, triggering downstream signaling. ROOT MERISTEM GROWTH FACTOR1 (RGF1) INSENSITIVEs (RGIs) LRR-RLK receptors promote primary root meristem activity while inhibiting lateral root (LR) development in response to RGF peptide. In this study, we employed rapamycin-induced dimerization (RiD) and BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) chimera approaches to explore the gain-of-function of RGI1, RGI4, and RGI5. Rapamycin induced the association of cytosolic kinase domains (CKDs) of RGI1 and the BAK1 coreceptor, activating both mitogen-activated protein kinase 3 (MPK3) and MPK6. Rapamycin significantly inhibited LR formation in RiD-RGI1/RGI4/RGI5-BAK1 plants. Using transgenic Arabidopsis expressing RGI1CKD fused to the BIR3-LRR chimera under estradiol control, we observed a substantial reduction in LR density upon ß-estradiol treatment. Additionally, we identified a decrease in root gravitropism in BIR3 chimera plants. In contrast, RiD-RGI/BAK1 plants did not exhibit defects in root gravitropism, implying the importance of combinatorial interactions between RGIs and SERK coreceptors in the inhibition of root gravitropism. Constitutive activation of RGIs with BAK1 in RiD-RGI/BAK1 plants by rapamycin treatment resulted in the inhibition of primary root growth, resembling the inhibitory effects observed with high concentrations of phytohormones on primary root elongation. Our findings highlight that the interactions between CKDs of RGIs and BAK1, constitutively induced by rapamycin or BIR3 chimera, efficiently control LR development.

ROOT MERISTEM GROWTH FACTOR1 (RGF1)-RGF1 INSENSITIVE 1 peptide-receptor pair inhibits lateral root development via the MPK6-PUCHI module in Arabidopsis.

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs) regulate primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade in Arabidopsis. However, it is unknown how RGF1 regulates lateral root (LR) development. Here, we show that the RGF1-RGI1 peptide-receptor pair negatively regulates LR development via activation of PUCHI encoding AP2/EREBP. Exogenous RGF1 peptides inhibited LR development of the wild type. However, the rgi1 mutants were partially or fully insensitive to RGF1 during LR development, whereas four other rgi single mutants, namely rgi2, rgi3, rgi4, and rgi5, were sensitive to RGF1 in inhibiting LR formation. Consistent with this, the red fluorescent protein (RFP) signals driven by the RGF1 promoter were detected at stage I and the following stages, overlapping with RGI1 expression. PUCHI expression was significantly up-regulated by RGF1 but completely inhibited in rgi1. LR development of puchi1-1 was insensitive to RGF1. PUCHI expression driven by the RGI1 promoter reduced LR density in both the wild type and rgi1,2,3. Further, mpk6, but not mpk3, displayed significantly down-regulated PUCHI expression and insensitive LR development in response to RGF1. Collectively, these results suggest that the RGF1-RGI1 module negatively regulates LR development by activating PUCHI expression via MPK6.

Recent advances in peptide signaling during Arabidopsis root development.

Roots provide the plant with water and nutrients and anchor it in a substrate. Root development is controlled by plant hormones and various sets of transcription factors. Recently, various small peptides and their cognate receptors have been identified as controlling root development. Small peptides bind to membrane-localized receptor-like kinases, inducing their dimerization with co-receptor proteins for signaling activation and giving rise to cellular signaling outputs. Small peptides function as local and long-distance signaling molecules involved in cell-to-cell communication networks, coordinating root development. In this review, we survey recent advances in the peptide ligand-mediated signaling pathways involved in the control of root development in Arabidopsis. We describe the interconnection between peptide signaling and conventional phytohormone signaling. Additionally, we discuss the diversity of identified peptide-receptor interactions during plant root development.

The Arabidopsis heterotrimeric G-protein ß subunit, AGB1, is required for guard cell calcium sensing and calcium-induced calcium release.

Cytosolic calcium concentration ([Ca2+ ]cyt ) and heterotrimeric G-proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao ) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gß subunit, AGB1, is required for four guard cell Cao responses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+ ]cyt oscillation; and inositol 1,4,5-trisphosphate (InsP3) production. Stomata in wild-type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Cao . By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double-mutants, as well as those of the agg1agg2 Gγ double-mutant, were insensitive to Cao . These behaviors contrast with ABA-regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G-protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus-specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6-detected [Ca2+ ]cyt oscillations in response to Cao , initiating only a single [Ca2+ ]cyt spike. Experimentally imposed [Ca2+ ]cyt oscillations restored stomatal closure in agb1. Yeast two-hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Cao induced InsP3 production in Col but not in agb1. In sum, G-protein signaling via AGB1/AGG1/AGG2 is essential for Cao -regulation of stomatal apertures, and stomatal movements in response to Cao apparently require Ca2+ -induced Ca2+ release that is likely dependent on Gßγ interaction with PLCs leading to InsP3 production.

A new discrete dynamic model of ABA-induced stomatal closure predicts key feedback loops.

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network's domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology.

The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis.

Stomata are the tiny valves on the plant surface that mediate gas exchange between the plant and its environment. Stomatal opening needs to be tightly regulated to facilitate CO2 uptake and prevent excess water loss. Plant Rho-type (ROP) GTPase 2 (ROP2) is a molecular component of the system that negatively regulates light-induced stomatal opening. Previously, ROP-interactive Cdc42- and Rac-interactive binding motif-containing protein 7 (RIC7) was suggested to function downstream of ROP2. However, the underlying molecular mechanism remains unknown. To understand the mechanism by which RIC7 regulates light-induced stomatal opening, we analyzed the stomatal responses of ric7 mutant Arabidopsis plants and identified the target protein of RIC7 using a yeast two-hybrid screen. Light-induced stomatal opening was promoted by ric7 knockout, whereas it was inhibited by RIC7 overexpression, indicating that RIC7 negatively regulates stomatal opening in Arabidopsis. RIC7 interacted with exocyst subunit Exo70 family protein B1 (Exo70B1), a component of the vesicle trafficking machinery. RIC7 and Exo70B1 localized to the plasma membrane region under light or constitutively active ROP2 conditions. The knockout mutant of Exo70B1 and ric7/exo70b1 exhibited retarded light-induced stomatal opening. Our results suggest that ROP2 and RIC7 suppress excess stomatal opening by inhibiting Exo70B1, which most likely participates in the vesicle trafficking required for light-induced stomatal opening.

Molecular and systems approaches towards drought-tolerant canola crops.

1169 I. 1170 II. 1170 III. 1172 IV. 1176 V. 1181 VI. 1182 1183 References 1183 SUMMARY: Modern agriculture is facing multiple challenges including the necessity for a substantial increase in production to meet the needs of a burgeoning human population. Water shortage is a deleterious consequence of both population growth and climate change and is one of the most severe factors limiting global crop productivity. Brassica species, particularly canola varieties, are cultivated worldwide for edible oil, animal feed, and biodiesel, and suffer dramatic yield loss upon drought stress. The recent release of the Brassica napus genome supplies essential genetic information to facilitate identification of drought-related genes and provides new information for agricultural improvement in this species. Here we summarize current knowledge regarding drought responses of canola, including physiological and -omics effects of drought. We further discuss knowledge gained through translational biology based on discoveries in the closely related reference species Arabidopsis thaliana and through genetic strategies such as genome-wide association studies and analysis of natural variation. Knowledge of drought tolerance/resistance responses in canola together with research outcomes arising from new technologies and methodologies will inform novel strategies for improvement of drought tolerance and yield in this and other important crop species.

Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants.

Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1.

Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells.

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K(+) and Ca(2+) currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K(+) channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca(2+) -permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K(+) channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.

The RGI1-BAK1 module acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs), a group of leucine-rich repeat receptor kinases, promote primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade and control root gravitropism in Arabidopsis. Genetic analyses and in vitro binding assays have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 recognize RGF1 peptides. However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in the regulation of primary root meristem activity. In the present study, we analyzed root meristem growth of the rgi1, rgi2, and rgi3 single mutants in response to RGF1 treatment and observed a significantly decreased sensitivity in meristem growth of rgi1 and complete insensitivity in rgi1 rgi2 rgi3 triple mutant compared with the wild type but not in the rgi1 and rgi2 single mutants. We also observed that both root gravitropism and meristem growth in the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (bak1) single mutant were insensitive to RGF1 peptide treatment, whereas other serk mutants, such as serk1, serk2, and serk4, were fully sensitive to RGF1 peptide like the wild type. These mutant analyses suggest that RGI1-BAK1 pair acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

Active ROP2 GTPase inhibits ABA- and CO2-induced stomatal closure.

ROP GTPases function as molecular switches in diverse cellular processes. Previously, we showed that ROP2 GTPase is activated upon light irradiation, and thereby negatively regulates light-induced stomatal opening. Here we studied the role of ROP2 during stomatal closure. The expression of a constitutively active form of ROP2 (CA-rop2) in Arabidopsis thaliana and Vicia faba resulted in slower and reduced stomatal closure in response to abscisic acid (ABA) and CO(2) . In contrast, the expression of a dominant-negative form of ROP2 (DN-rop2) and the knockout mutation of ROP2 (rop2 KO) promoted ABA-induced stomatal closure in Arabidopsis. As early as 10 min after ABA treatment, ROP2 was inactivated and translocated to the cytoplasm of the stomatal guard cells. To elucidate the mechanism by which active ROP2 suppresses stomatal closure, we monitored endocytotic membrane trafficking, which is regulated by Rho GTPases in animal cells. We found that the endocytosis of plasma membrane (PM), as tracked by FM4-64, was lower in CA-rop2-expressing guard cells than in those of wild-type plants, which suggests that active ROP2 suppresses the endocytotic internalization of PM, a process required for stomatal closure. Together, our results suggest that ROP2 is inactivated by ABA, and that this inactivation is required for the timely stomatal closure.

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells.

Heterotrimeric G proteins composed of Gα, Gß, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.

Signaling Peptides Regulating Abiotic Stress Responses in Plants.

As sessile organisms, plants are exposed to constantly changing environments that are often stressful for their growth and development. To cope with these stresses, plants have evolved complex and sophisticated stress-responsive signaling pathways regulating the expression of transcription factors and biosynthesis of osmolytes that confer tolerance to plants. Signaling peptides acting like phytohormones control various aspects of plant growth and development via cell-cell communication networks. These peptides are typically recognized by membrane-embedded receptor-like kinases, inducing activation of cellular signaling to control plant growth and development. Recent studies have revealed that several signaling peptides play important roles in plant responses to abiotic stress. In this mini review, we provide recent findings on the roles and signaling pathways of peptides that are involved in coordinating plant responses to abiotic stresses, such as dehydration, high salinity, reactive oxygen species, and heat. We also discuss recent developments in signaling peptides that play a role in plant adaptation responses to nutrient deficiency stress, focusing on nitrogen and phosphate deficiency responses.

Peptide Signaling during Plant Reproduction.

Plant signaling peptides are involved in cell-cell communication networks and coordinate a wide range of plant growth and developmental processes. Signaling peptides generally bind to receptor-like kinases, inducing their dimerization with co-receptors for signaling activation to trigger cellular signaling and biological responses. Fertilization is an important life event in flowering plants, involving precise control of cell-cell communications between male and female tissues. Peptide-receptor-like kinase-mediated signaling plays an important role in male-female interactions for successful fertilization in flowering plants. Here, we describe the recent findings on the functions and signaling pathways of peptides and receptors involved in plant reproduction processes including pollen germination, pollen tube growth, pollen tube guidance to the embryo sac, and sperm cell reception in female tissues.

Chemical control of receptor kinase signaling by rapamycin-induced dimerization.

Membrane-localized leucine-rich repeat receptor kinases (LRR-RKs) sense diverse extracellular signals, and coordinate and specify cellular functions in plants. However, functional understanding and identification of the cellular signaling of most LRR-RKs remain a major challenge owing to their genetic redundancy, the lack of ligand information, and subtle phenotypes of LRR-RK overexpression. Here, we report an engineered rapamycin-inducible dimerization (RiD) receptor system that triggers a receptor-specific LRR-RK signaling independent of their cognate ligands or endogenous receptors. Using the RiD-receptors, we demonstrated that the rapamycin-mediated association of chimeric cytosolic kinase domains from the BRI1/BAK1 receptor/co-receptor, but not the BRI1/BRI1 or BAK1/BAK1 homodimer, is sufficient to activate downstream brassinosteroid signaling and physiological responses. Furthermore, we showed that the engineered RiD-FLS2/BAK1 could activate flagellin-22-mediated immune signaling and responses. Using the RiD system, we also identified the potential function of an unknown orphan receptor in immune signaling and revealed the differential activities of SERK co-receptors of LRR-RKs. Our results indicate that the RiD method can serve as a synthetic biology tool for precise temporal manipulation of LRR-RK signaling and for understanding LRR-RK biology.

Arabidopsis annexins AnnAt1 and AnnAt4 interact with each other and regulate drought and salt stress responses.

Annexins are Ca2+--and phospholipid-binding proteins that form an evolutionarily conserved multigene family throughout the animal and plant kingdoms. Two annexins, AnnAt1 and AnnAt4, have been identified as components in osmotic stress and abscisic acid signaling in Arabidopsis. Here, we report that AnnAt1 and AnnAt4 regulate plant stress responses in a light-dependent manner. The single-mutant annAt1 and annAt4 plants showed tolerance to drought and salt stress, which was greatly enhanced in double-mutant annAt1annAt4 plants, but AnnAt4-overexpressing transgenic plants (35S:AnnAt4) were more sensitive to stress treatments under long day conditions. Furthermore, expression of stress-related genes was altered in these mutant and transgenic plants. Upon dehydration and salt treatment, AtNCED3, encoding 9-cis-epoxycarotenoid dioxygenase, and P5CS1, encoding Δ-1-pyrroline-5-carboxylate synthase, which are key enzymes in ABA and proline synthesis, respectively, were highly induced in annAt1annAt4 plants and to a lesser extent in annAt1 and annAt4 plants, but not in 35S:AnnAt4 plants. While annAt1 plants were more drought sensitive, annAt4 plants were more tolerant in short days than in long days. In vitro and in vivo binding assays revealed that AnnAt1 and AnnAt4 bind to each other in a Ca2+-dependent manner. Our results suggest that AnnAt1 and AnnAt4 function cooperatively in response to drought and salt stress and their functions are affected by photoperiod.

Phosphatidylinositol 3- and 4-phosphate modulate actin filament reorganization in guard cells of day flower.

Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P(3)) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P(3) nor the enzyme that produces this lipid has been reported. However, a PtdIns 3-kinase that produces phosphatidylinositol 3-phosphate (PtdIns3P) has been identified, suggesting that PtdIns3P, instead of PtdIns(3,4,5)P(3), regulates actin dynamics in plant cells. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, we investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower (Commelina communis). ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, we demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.

Role of LBD14 during ABA-mediated control of root system architecture in Arabidopsis.

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encode plant-specific transcription factors that regulate various aspects of plant growth and development. Arabidopsis genome has 42 LBD genes. Several LBD genes, such as LBD16, -18, -29, and -33, have been shown to function in lateral root (LR) development via auxin signaling. Although abscisic acid (ABA) is a well-known antistress plant hormone regulating various plant developmental processes, it also plays a role in LR growth regulation. Our recent study showed that LBD14 expression is downregulated by ABA during the entire steps of LR development. The RNAi-induced downregulation and overexpression of LBD14 indicated that LBD14 promotes LR formation. LBD14RNAi enhanced the ABA-induced suppression of LR density compared with the wild type, suggesting that LBD14 is involved in the ABA-mediated control of LR formation. Our study provides an insight into the signaling mechanism of developmental plasticity whereby ABA controls LR branching via LBD14 downregulation under abiotic stress conditions.

Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function.

Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae.

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.