Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways.

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

SummaryThe blue-light-induced trafficking of the UVA/blue light receptor phototropin2 is shown. Evidence is provided for the presence of two pathways, one directing phototropin2 to the Golgi and post-Golgi vesicles, and the other to degradation.

Abstract:The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The OPEN ACCESSMolecules 2014, 19 13393 activity of β-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.

The EGY3 protein is a homologue of site‐2 proteases, which are intramembrane zinc metalloproteases. EGY3 itself lacks proteolytic activity due to the absence of a zinc‐binding motif. Plentiful evidence indicates that such intramembrane ‘pseudoproteases’ play significant roles in many diverse processes occurring within the cell. However, the physiological functions of EGY3, as well as its subcellular localization, remain unknown. The subcellular localization of EGY3 protein was investigated using Arabidopsis thaliana protoplasts transformed with EGY3‐GFP fusion protein, and immunoblot experiments using the total leaf protein extract, as well as highly purified chloroplasts and fractions of stroma, envelope and thylakoid membrane proteins. The physiological role of EGY3 was studied using two A. thaliana mutant lines devoid of EGY3 protein. Chlorophyll a fluorescence measurement was performed and the egy3 mutant sensitivity to photoinhibition was investigated. Additionally, the abundance of thylakoid membrane complexes was established using blue native gel electrophoresis. We present experimental evidence for thylakoid membrane localization of the EGY3 protein. We show that egy3 mutants display increased value of the non‐photochemical quenching parameter and significantly slower recovery rate after photoinhibitory treatment. This was associated with a decrease in the level of proteases involved in photosystem II recovery, Deg1 and FtsH2/8.

Summary In trees, dead and living cells of secondary xylem (wood) function collectively, rendering cell‐to‐cell communication challenging. Water and solutes are transported over long distances from the roots to the above‐ground organs via vessels, the main component of wood, and then radially over short distances to the neighboring cells. This enables proper functioning of trees and integrates whole‐plant activity. In this study, tracer loading, immunolocalization experiments and inhibitor assays were used to decipher the mechanisms enabling transport in wood of Acer pseudoplatanus (maple), Fraxinus excelsior (ash) and Populus tremula × tremuloides (poplar) trees. We show that tracer uptake from dead water‐conducting vessels, elements of the apoplasm, to living vessel‐associated cells (VACs) of the xylem parenchyma of the symplasm system proceeds via the endocytic pathway, including clathrin‐mediated and clathrin‐independent processes. These findings enhance our understanding of the transport pathways in complex wood tissue, providing experimental evidence of the involvement of VACs and endocytosis in radial uptake from vessels.

Ethylene is an important plant hormone that controls growth, development, aging and stress responses. The rate-limiting enzymes in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylate synthases (ACSs), are strictly regulated at many levels, including posttranslational control of protein half-life. Reversible phosphorylation/dephosphorylation events play a pivotal role as signals for ubiquitin-dependent degradation. We showed previously that ABI1, a group A protein phosphatase type 2C (PP2C) and a key negative regulator of abscisic acid signaling regulates type I ACS stability. Here we provide evidence that ABI1 also contributes to the regulation of ethylene biosynthesis via ACS7, a type III ACS without known regulatory domains. Using various approaches, we show that ACS7 interacts with ABI1, ABI2 and HAB1. We use molecular modeling to predict the amino acid residues involved in ABI1/ACS7 complex formation and confirm these predictions by mcBiFC-FRET-FLIM analysis. Using a cell-free degradation assay, we show that proteasomal degradation of ACS7 is delayed in protein extracts prepared from PP2C type A knockout plants, compared to a wild-type extract. This study therefore shows that ACS7 undergoes complex regulation governed by ABI1, ABI2 and HAB1. Furthermore, this suggests that ACS7, together with PP2Cs, plays an essential role in maintaining appropriate levels of ethylene in Arabidopsis.Cells 2020, 9, 978 2 of 20 also abbreviated as ACC synthase) to 1-aminocyclopropane 1-carboxylate (ACC). In the second step, ACC is converted to ethylene with release of carbon dioxide and cyanide by 1-aminocyclopropane 1-carboxylate oxidase (ACO). ACS is thus a crucial enzyme in ethylene biosynthesis and in all plant species is encoded by a multigene family consisting of nine members in Lycopersicon esculentum, six in Oriza sativa and 11 in poplar [7,8]. In Arabidopsis, the ACS gene family comprises 12 members, including eight genes encoding functional enzymes (ACS2, ACS4-9 and ACS11), a single inactive form of ACS1 and a pseudogene ACS3 [9].The available domain structures of the ACS isozymes allow their classification based on the presence or absence of non-catalytic C-terminal phosphorylation motifs [10]. Type I ACSs contain a C-terminal fragment that includes phosphorylation sites for both mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs). In Arabidopsis thaliana, ACS2 and ACS6 belong to type I. Type II isozymes carry only the CDPK target site, and type III isozymes lack both MAPK and CDPK sites [11,12]. In Arabidopsis, ACS4, ACS5, ACS8, ACS9 and ACS11 are type II ACSs, while there is only a single type III isozyme, ACS7.Although ACS7 lacks known regulatory domains, it still undergoes ubiquitin-dependent proteasomal degradation. The E3 ubiquitin ligase, XBAT32, initiates this process by attachment of ubiquitin to ACS7 [13,14]. XBAT32 is member of the RING domain-containing ankyrin repeat E3 ligase subfamily and was first characterized as a positive regulator of lateral root develo...