STOP1-regulated SMALL AUXIN UP RNA55 (SAUR55) is involved in proton/malate co-secretion for Al tolerance in Arabidopsis.

Proton (H+) release is linked to aluminum (Al)-enhanced organic acids (OAs) excretion from the roots under Al rhizotoxicity in plants. It is well-reported that the Al-enhanced organic acid excretion mechanism is regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1), a zinc-finger TF that regulates major Al tolerance genes. However, the mechanism of H+ release linked to OAs excretion under Al stress has not been fully elucidated. Recent physiological and molecular-genetic studies have implicated the involvement of SMALL AUXIN UP RNAs (SAURs) in the activation of plasma membrane H+-ATPases for stress responses in plants. We hypothesized that STOP1 is involved in the regulation of Al-responsive SAURs, which may contribute to the co-secretion of protons and malate under Al stress conditions. In our transcriptome analysis of the roots of the stop1 (sensitive to proton rhizotoxicity1) mutant, we found that STOP1 regulates the transcription of one of the SAURs, namely SAUR55. Furthermore, we observed that the expression of SAUR55 was induced by Al and repressed in the STOP1 T-DNA insertion knockout (KO) mutant (STOP1-KO). Through in silico analysis, we identified a functional STOP1-binding site in the promoter of SAUR55. Subsequent in vitro and in vivo studies confirmed that STOP1 directly binds to the promoter of SAUR55. This suggests that STOP1 directly regulates the expression of SAUR55 under Al stress. We next examined proton release in the rhizosphere and malate excretion in the T-DNA insertion KO mutant of SAUR55 (saur55), in conjunction with STOP1-KO. Both saur55 and STOP1-KO suppressed rhizosphere acidification and malate release under Al stress. Additionally, the root growth of saur55 was sensitive to Al-containing media. In contrast, the overexpressed line of SAUR55 enhanced rhizosphere acidification and malate release, leading to increased Al tolerance. These associations with Al tolerance were also observed in natural variations of Arabidopsis. These findings demonstrate that transcriptional regulation of SAUR55 by STOP1 positively regulates H+ excretion via PM H+-ATPase 2 which enhances Al tolerance by malate secretion from the roots of Arabidopsis. The activation of PM H+-ATPase 2 by SAUR55 was suggested to be due to PP2C.D2/D5 inhibition by interaction on the plasma membrane with its phosphatase. Furthermore, RNAi-suppression of NtSTOP1 in tobacco shows suppression of rhizosphere acidification under Al stress, which was associated with the suppression of SAUR55 orthologs, which are inducible by Al in tobacco. It suggests that transcriptional regulation of Al-inducible SAURs by STOP1 plays a critical role in OAs excretion in several plant species as an Al tolerance mechanism.

Discovery and Genome Characterization of a Closterovirus from Wheat Plants with Yellowing Leaf Symptoms in Japan.

Many aphid-borne viruses are important pathogens that affect wheat crops worldwide. An aphid-transmitted closterovirus named wheat yellow leaf virus (WYLV) was found to have infected wheat plants in Japan in the 1970s; however, since then, its viral genome sequence and occurrence in the field have not been investigated. We observed yellowing leaves in the 2018/2019 winter wheat-growing season in an experimental field in Japan where WYLV was detected five decades ago. A virome analysis of those yellow leaf samples lead to the discovery of a closterovirus together with a luteovirus (barley yellow dwarf virus PAV variant IIIa). The complete genomic sequence of this closterovirus, named wheat closterovirus 1 isolate WL19a (WhCV1-WL19a), consisted of 15,452 nucleotides harboring nine open reading frames. Additionally, we identified another WhCV1 isolate, WL20, in a wheat sample from the winter wheat-growing season of 2019/2020. A transmission test indicated that WhCV1-WL20 was able to form typical filamentous particles and transmissible by oat bird-cherry aphid (Rhopalosiphum pad). Sequence and phylogenetic analyses showed that WhCV1 was distantly related to members of the genus Closterovirus (family Closteroviridae), suggesting that the virus represents a novel species in the genus. Furthermore, the characterization of WhCV1-WL19a-derived small RNAs using high-throughput sequencing revealed highly abundant 22-nt-class small RNAs potentially derived from the 3'-terminal end of the WhCV1 negative-strand genomic RNA, indicating that this terminal end of the WhCV1 genome is likely particularly targeted for the synthesis of viral small RNAs in wheat plants. Our results provide further knowledge on closterovirus diversity and pathogenicity and suggest that the impact of WhCV1 on wheat production warrants further investigations.

Misplacement of left ventricular vent into the aortic root during a re-do Bentall procedure: a case report.

BACKGROUND: The left ventricular (LV) vent is commonly inserted via the right superior pulmonary vein (RSPV) and directed toward the LV cavity through the mitral valve. We report a rare case in which the tip of the LV vent was misplaced into the aortic root across the aortic valve. CASE PRESENTATION: An 88-year-old man was scheduled to undergo the Bentall procedure. After initiation of cardiopulmonary bypass, the LV vent was inserted via the RSPV. Anterograde cardioplegia was administered via the aortic root cannula after the ascending aorta was cross-clamped. The electrocardiogram did not result in complete cardiac arrest, even after delivery of two-thirds of the planned dose. A transesophageal echocardiographic examination showed that the tip of the LV vent was misplaced into the aortic root across the aortic valve. CONCLUSIONS: It is important to confirm the tip position by transesophageal echocardiography to prevent severe complications associated with the LV vent.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Inter-tissue and inter-organ signaling in drought stress response and phenotyping of drought tolerance.

Plant response to drought stress includes systems for intracellular regulation of gene expression and signaling, as well as inter-tissue and inter-organ signaling, which helps entire plants acquire stress resistance. Plants sense water-deficit conditions both via the stomata of leaves and roots, and transfer water-deficit signals from roots to shoots via inter-organ signaling. Abscisic acid is an important phytohormone involved in the drought stress response and adaptation, and is synthesized mainly in vascular tissues and guard cells of leaves. In leaves, stress-induced abscisic acid is distributed to various tissues by transporters, which activates stomatal closure and expression of stress-related genes to acquire drought stress resistance. Moreover, the stepwise stress response at the whole-plant level is important for proper understanding of the physiological response to drought conditions. Drought stress is sensed by multiple types of sensors as molecular patterns of abiotic stress signals, which are transmitted via separate parallel signaling networks to induce downstream responses, including stomatal closure and synthesis of stress-related proteins and metabolites. Peptide molecules play important roles in the inter-organ signaling of dehydration from roots to shoots, as well as signaling of osmotic changes and reactive oxygen species/Ca2+ . In this review, we have summarized recent advances in research on complex plant drought stress responses, focusing on inter-tissue signaling in leaves and inter-organ signaling from roots to shoots. We have discussed the mechanisms via which drought stress adaptations and resistance are acquired at the whole-plant level, and have proposed the importance of quantitative phenotyping for measuring plant growth under drought conditions.

Identification of a Novel Quinvirus in the Family Betaflexiviridae That Infects Winter Wheat.

Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation.

High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis.

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis.

CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice.

Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the ß-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.

The genotype-dependent phenotypic landscape of quinoa in salt tolerance and key growth traits.

Cultivation of quinoa (Chenopodium quinoa), an annual pseudocereal crop that originated in the Andes, is spreading globally. Because quinoa is highly nutritious and resistant to multiple abiotic stresses, it is emerging as a valuable crop to provide food and nutrition security worldwide. However, molecular analyses have been hindered by the genetic heterogeneity resulting from partial outcrossing. In this study, we generated 136 inbred quinoa lines as a basis for the molecular identification and characterization of gene functions in quinoa through genotyping and phenotyping. Following genotyping-by-sequencing analysis of the inbred lines, we selected 5,753 single-nucleotide polymorphisms (SNPs) in the quinoa genome. Based on these SNPs, we show that our quinoa inbred lines fall into three genetic sub-populations. Moreover, we measured phenotypes, such as salt tolerance and key growth traits in the inbred quinoa lines and generated a heatmap that provides a succinct overview of the genotype-phenotype relationship between inbred quinoa lines. We also demonstrate that, in contrast to northern highland lines, most lowland and southern highland lines can germinate even under high salinity conditions. These findings provide a basis for the molecular elucidation and genetic improvement of quinoa and improve our understanding of the evolutionary process underlying quinoa domestication.

Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses.

Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to -4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to -4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

Cellulose-rich secondary walls in wave-swept red macroalgae fortify flexible tissues.

MAIN CONCLUSION: Cellulosic secondary walls evolved convergently in coralline red macroalgae, reinforcing tissues against wave-induced breakage, despite differences in cellulose abundance, microfibril orientation, and wall structure. Cellulose-enriched secondary cell walls are the hallmark of woody vascular plants, which develop thickened walls to support upright growth and resist toppling in terrestrial environments. Here we investigate the striking presence and convergent evolution of cellulosic secondary walls in coralline red algae, which reinforce thalli against forces applied by crashing waves. Despite ostensible similarities to secondary wall synthesis in land plants, we note several structural and mechanical differences. In coralline red algae, secondary walls contain three-times more cellulose (~ 22% w/w) than primary walls (~ 8% w/w), and their presence nearly doubles the total thickness of cell walls (~ 1.2 µm thick). Field emission scanning electron microscopy revealed that cellulose bundles are cylindrical and lack any predominant orientation in both primary and secondary walls. His-tagged recombinant carbohydrate-binding module differentiated crystalline and amorphous cellulose in planta, noting elevated levels of crystalline cellulose in secondary walls. With the addition of secondary cell walls, Calliarthron genicular tissues become significantly stronger and tougher, yet remain remarkably extensible, more than doubling in length before breaking under tension. Thus, the development of secondary walls contributes to the strong-yet-flexible genicular tissues that enable coralline red algae to survive along wave-battered coastlines throughout the NE Pacific. This study provides an important evolutionary perspective on the development and biomechanical significance of secondary cell walls in a non-model, non-vascular plant.

Extracellular Spermine Triggers a Rapid Intracellular Phosphatidic Acid Response in Arabidopsis, Involving PLDδ Activation and Stimulating Ion Flux.

Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low µM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential 32Pi-labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular PLDδ was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and pldδ-mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K+. Potential mechanisms of how PLDδ and the increase of PA are involved in polyamine function is discussed.

Two novel fungal negative-strand RNA viruses related to mymonaviruses and phenuiviruses in the shiitake mushroom (Lentinula edodes).

There is still limited information on the diversity of (-)ssRNA viruses that infect fungi. Here, we have discovered two novel (-)ssRNA mycoviruses in the shiitake mushroom (Lentinula edodes). The first virus has a monopartite RNA genome and relates to that of mymonaviruses (Mononegavirales), especially to Hubei rhabdo-like virus 4 from arthropods and thus designated as Lentinula edodes negative-strand RNA virus 1. The second virus has a putative bipartite RNA genome and is related to the recently discovered bipartite or tripartite phenui-like viruses (Bunyavirales) associated with plants and ticks, and designated as Lentinula edodes negative-strand RNA virus 2 (LeNSRV2). LeNSRV2 is likely the first segmented (-)ssRNA virus known to infect fungi. Its smaller RNA segment encodes a putative nucleocapsid and a plant MP-like protein using a potential ambisense coding strategy. These findings enhance our understanding of the diversity, evolution and spread of (-)ssRNA viruses in fungi.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

Correction to: Casein kinase 2 α and ß subunits inversely modulate ABA signal output in Arabidopsis protoplasts.

The article Casein kinase 2 α and ß subunits inversely modulate ABA signal output in Arabidopsis protoplasts, written by Yukari Nagatoshi, Miki Fujita, and Yasunari Fujita, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 24 May 2018 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 19 November 2018 to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

RIPPS: A Plant Phenotyping System for Quantitative Evaluation of Growth Under Controlled Environmental Stress Conditions.

High-throughput and accurate measurements of plant traits facilitate identification of gene function. Along with recent advances in quantitative genomics, there is a growing need for precise quantification of multiple traits in plants. However, it is difficult continuously to quantify plant adaptive responses to environmental stress responses such as drought because multiple environmental factors are intricately involved in the phenotype. To solve this problem, we developed an automatic phenotyping system for evaluating the growth responses of individual Arabidopsis plants to a wide range of environmental conditions. The RIKEN Integrated Plant Phenotyping System (RIPPS) controls soil moisture for single plants by automatically weighing and watering 120 continuously rotating pots under controlled light, humidity and temperature growth conditions. RIPPS also records individual rosette size and expansion rate by photographing plants every 2 h. We used RIPPS to establish phenotype evaluation methods for Arabidopsis growth response and water use efficiency under various water conditions, and analyzed the involvement of ABA metabolism in determining water use efficiency. We also used RIPPS to analyze salinity tolerance in Arabidopsis plants.

Exploring Microtubule-Dependent Cellulose-Synthase-Complex Movement with High Precision Particle Tracking.

Cellulose synthesis at the plasma membrane is a critical process in plant growth and development. The displacement of cellulose synthase complexes (CSCs) by the rigid cellulose polymers they produce is a measure of enzyme activity. Connections between cortical microtubules and CSCs have been identified but it remains unclear how these affect CSC displacement speed. In this study, we applied a high throughput automated particle tracking method using near-total internal reflection fluorescence microscopy to measure the speed of CSCs. We found CSC speeds did not vary according to their proximity to microtubules, and that inhibiting microtubule polymerization could have opposite effects on CSC speed, depending on the nature of inhibition. While CSC speed increased in the temperature-sensitive mor1-1 mutant, it decreased after treatment with the drug oryzalin. Moreover, introducing the mor1-1 mutation into the CesA1 mutant any1 increased CSC speed, suggesting that microtubule dynamics affect CSC speed by a mechanism other than Cellulose Synthase A (CesA) catalytic activity. CSC speed varied widely in a range of mutants with reduced growth anisotropy, indicating that the relationship between CSC speed and anisotropy is complex. We conclude that microtubules affect CSC speed by finely tuned mechanisms that are independent of their physical association with CSCs.

Casein kinase 2 α and ß subunits inversely modulate ABA signal output in Arabidopsis protoplasts.

MAIN CONCLUSION: Our transient gene expression analyses in Arabidopsis protoplasts support the view that CK2αs and CK2ßs positively and negatively modulate ABRE-dependent gene expression, respectively. The phytohormone abscisic acid (ABA) regulates the expression of thousands of genes via ABA-responsive elements (ABREs), and has a crucial role in abiotic stress response. Casein kinase II (CK2), a conserved Ser/Thr protein kinase in eukaryotes, is essential for plant viability. Although the CK2 has been known as a tetrameric holoenzyme comprised of two catalytic α and two regulatory ß subunits, each of the two types of subunits has been proposed to have independent functions. The Arabidopsis genome encodes four α subunits (CK2α1, CK2α2, CK2α3, CK2α4) and four ß subunits (CK2ß1, CK2ß2, CK2ß3, CK2ß4). There is a growing body of evidence linking CK2 to ABA signaling and abiotic stress responses. However, the roles of each CK2 subunit in ABA signaling remain largely elusive. Using the transient expression system with the core ABA signaling components in Arabidopsis leaf mesophyll protoplasts, we show here that CK2α1 and CK2α2 (CK2α1/2) positively modulate ABRE-dependent gene expression as ABA signal output in ABA signaling, whereas all four CK2ßs negatively modulate the ABRE-dependent gene expression mediated by subclass III SnRK2-AREB/ABF pathway and by CK2α1/2. These data indicate that CK2α1/2 and CK2ßs positively and negatively modulate ABA signal output, respectively, suggesting that the quantitative balance of CK2 subunits determines the ABA signal output in plants. Given that CK2s act as pleiotropic enzymes involved in multiple developmental and stress-responsive processes, our findings suggest that CK2 subunits may be involved in integration and coordination of ABA-dependent and -independent signaling.

Overexpression of AtABCG25 enhances the abscisic acid signal in guard cells and improves plant water use efficiency.

In addition to improving drought tolerance, improvement of water use efficiency is a major challenge in plant physiology. Due to their trade-off relationships, it is generally considered that achieving stress tolerance is incompatible with maintaining stable growth. Abscisic acid (ABA) is a key phytohormone that regulates the balance between intrinsic growth and environmental responses. Previously, we identified AtABCG25 as a cell-membrane ABA transporter that export ABA from the inside to the outside of cells. AtABCG25-overexpressing plants showed a lower transpiration phenotype without any growth retardation. Here, we dissected this useful trait using precise phenotyping approaches. AtABCG25 overexpression stimulated a local ABA response in guard cells. Furthermore, AtABCG25 overexpression enhanced drought tolerance, probably resulting from maintenance of water contents over the common threshold for survival after drought stress treatment. Finally, we observed enhanced water use efficiency by overexpression of AtABCG25, in addition to drought tolerance. These results were consistent with the function of AtABCG25 as an ABA efflux transporter. This unique trait may be generally useful for improving the water use efficiency and drought tolerance of plants.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.