SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation

Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms.In strigolactone signaling,the enzyme receptor DWARF14(D14)and an F-box protein,MORE AXILLARY GROWTH2(MAX2),mark SUPPRESSOR OF MAX21-LIKE(SMXL)family proteins SMXL6,SMXL7,and SMXL8 for rapid degradation.Removal of these transcriptional corepressors initiates downstream growth responses.The homologous proteins SMXL3,SMXL4,and SMXL5,however,are resistant to MAX2-mediated degradation.We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone.SMXL5 attenuates strigolactone signaling by interfering with AtD14-SMXL7 interactions.SMXL5 interacts with AtD14 and SMXL7,providing two possible ways to inhibit SMXL7 degradation.SMXL5 function is partially dependent on an ethylene-responsive-element binding-factor-associated amphiphilic repression(EAR)motif,which typically mediates interactions with the TOPLESS family of transcriptional corepressors.However,we found that loss of the EAR motif reduces SMXL5-SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5.We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation and that the EAR motif promotes the formation or stability of these complexes.This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation.

Epigenetic Modifications of mRNA and DNA in Plants

Advances in the detection and mapping of messenger RNA(mRNA)N^6-methyladenosine(m 6A)and 5-methylcytosine(m 5C),and DNA N^6-methyldeoxyadenosine(6mA)redefined our understanding of these modifications as additional tiers of epigenetic regulation.In plants,the most prevalent internal mRNA modifications,m^6A and m^5C,play crucial and dynamic roles in many processes,including embryo development,stem cell fate determination,trichome branching,leaf morphogenesis,floral transition,stress responses,fruit ripening,and root development.The newly identified and widespread epigenetic marker 6mA DNA methylation is associated with gene expression,plant development,and stress responses.Here,we review the latest research progress on mRNA and DNA epigenetic modifications,including the detection,dynamics,distribution,functions,regulatory proteins,and evolution,with a focus on m^6A,m^5C,and 6mA.We also provide some perspectives on future research of the newly identified and unknown epigenetic modifications of mRNA and DNA in plants.

Transcriptional landscape of rice roots at the single-cell resolution

There are two main types of root systems in flowering plants,namely taproot systems of dicots and fibrous root systems found in monocots.Despite this fundamental split,our current knowledge of cellular and molecular mechanism driving root development is mainly based on studies of the dicot model Arabidopsis.However,the world major crops are monocots and little is known about the transcriptional programs underlying cell-type specification in this clade.Here,we report the transcriptomes of more than 20000 single cells derived from root tips of two agronomically important rice cultivars.Using combined computational and experimental analyses we were able to robustly identify most of the major cell types and define novel cell-type-specific marker genes for both cultivars.Importantly,we found divergent cell types associated with specific regulatory programs,including phytohormone biosynthesis,signaling,and response,which were well conserved between the two rice cultivars.In addition,we detected substantial differences between the cell-type transcript profiles of Arabidopsis and rice.These species-specific features emphasize the importance of analyzing tissues across diverse model species,including rice.Taken together,our study provides insight into the transcriptomic landscape of major cell types of rice root tip at single-cell resolution and opens new avenues to study cell-type specification,function,and evolution in plants.

Live Cell Imaging with R-GEC01 Sheds Light on fig22- and Chitin-Induced Transient [Ca2＋]cyt Patterns in Arabidopsis

Intracellular Ca2＋ transients are an integral part of the signaling cascade during pathogen-associated molecular pattern （PAMP）-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca2＋ signals is limited. Investigation of cell- and tissue-specific properties of Ca2＋- dependent signaling processes requires versatile Ca2＋ reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca2＋ signaling in living cells. In this study, we compared two fluorescence-based Ca2＋ sensors： the F6rster resonance energy transfer （FRET）-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report fig22- and chitin-induced Ca2＋ signals on a cellular scale, which allowed identification of defined [Ca2＋]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that fig22- and chitin-induced Ca2＋ signals in the root initiate from the elongation zone.

Regulation of the New Arabidopsis Imprinted Gene AtBMIIC Requires the Interplay of Different Epigenetic Mechanisms

Recently, it has been shown that plants contain homologs to the animal Polycomb repressive complex I （PRC1） components BM11 and RINGIA/B. In Arabidopsis, there are three BMIl-like genes, two of which, AtBMIIA and B, are required during post-embryonic plant growth to repress embryonic traits and allow cell differentiation. However, little is known about the third BMIl-like gene, AtBMIIC. In this work, we show that AtBMIIC is only expressed during endosperm and stamen development. AtBMIIC is an imprinted gene expressed from the maternal allele in the endosperm but bialleli- cally expressed in stamen. We found that the characteristic expression pattern of AtBMIIC is the result of a complex epigenetic regulation that involves CG DNA methylation, RNA-directed non-CG DNA methylation （RdDM）, and PcG activity. Our results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

OsHYPK-mediated protein N-terminal acetylation coordinates plant development and abiotic stress responses in rice

N-terminal acetylation is one of the most common protein modifications in eukaryotes,and approximately 40%of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A(NatA)in a co-translational manner.However,the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain unclear.Here,we identify Huntingtin Yeast partner K(HYPK),an evolutionarily conserved chaperone-like protein,as a positive regulator of NatA activity in rice.We found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses,attributable to perturbation of the N-terminal acetylome and accelerated global protein turnover.Furthermore,we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation,probably through the Ac/N-degron pathway,which could be induced by abiotic stresses.Taken together,our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover,which are essential for maintaining adaptive phenotypic plasticity in rice.

Specifying the role of BAK1-interacting receptor-like kinase 3 in brassinosteroid signaling

Brassinosteroids(BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1-associated kinase 1(BAK1)-interacting receptor-like kinase 3(BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1(BRI1) and BAK1.However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 Rewith BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.

Cytosolic Ca^2＋ Signals Enhance the Vacuolar Ion Conductivity of Bulging Arabidopsis Root Hair Cells

Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.

On-Site Manufacturing in Tip-Growing Cells through RALF1-FERONIA-Mediated Local mRNA Translation

Construction sites,particularly in the public sector,are notoriously unpredictable.No matter how well thought out the construction plan,no matter how rigorously calculated the time schedule,the rate of construction or the time point when the building will ultimately welcome its residents remains largely,volatile.The availability of resources,interactions between contractors,and environmental conditions,as well as control mechanisms and restrictions,form a complex"regulatory network"that governs the construction progress largely independent of the architecfs schedule and accounts for a certain"plasticity"along the way.

Endocytosis： Is There Really a Recycling from Late Endosomes？

Dear Editor,Over the last 15 years endocytosis has moved from being a process of only minor importance to plant physiologists to being one of the most exciting research areas in plant cell biology. These days, nobody doubts the operation of clathrin-mediated endocytosis as a mechanism for the internalization of a range of physiologically important transmembrane protein complexes at the plasma membrane （PM） of plant cells. These include both receptors and transporters. As in animal cells, most of these proteins are constitutively recycled back to the PM from an early endosome （EE）. However, some are destined for degradation and proceed further downstream in the endocytic pathway to late endosomes （LE）, where they are internalized into the intraluminal vesicles of the LE. Fusion of the LE with the lysosome/vacuole releases the vesicles leading to their degradation. The signal that marks PM proteins for degradation has been known in mammalian and yeast cells for quite some time and is polyubiquitination （Mukhopadhyay and Riezman, 2007）. In their Spotlight article, Zelazny and Vert （2015） highlight recent publications from the plant field that also demonstrate a key role for multiple monoubiquitination in the endocytosis of the metal transporters IRT1 （Barberon et al., 2011）, BOR1 （Kasai et al., 2011）, as well as lysine63-1inked polyubiquitination in vacuolar sorting of the auxin transporter PIN2 （Leitner et al., 2012）. They also draw attention to the discovery of a ubiquitin-binding protein （FREE1 ; also termed FYVE1 by Barberon et al., 2014） which locates to the LE in plant cells and is part of the ESCRT-I （endosomal sorting complex for transport） complex （Gao et al., 2014）. This complex sequesters ubiquitinated membrane cargo proteins and internalizes them. Finally, Zelazny and Vert draw attention to the work of Ivanov et al. （2014） in showing an increase in IRT1 degradation and iron deficiency in SNX1 mutants. So far so good, but Zelazny and Vert go on to conclude that a portion of the internalized IRT1 molecules that reach the LE are recycled back to the EE, a process that they consider to be mediated by sorting nexin 1 （SNX1）, a retromer protein. I question the soundness of this scenario.