Arabidopsis Protein Research Articles

Insights into the genes involved in the ethylene biosynthesis pathway in Arabidopsis thaliana and Oryza sativa

BackgroundEthylene is a gaseous plant hormone that acts as a requisite role in many aspects of the plant life cycle, and it is also a regulator of plant responses to abiotic and biotic stresses. In this study, we attempt to provide comprehensive information through analyses of existing data using bioinformatics tools to compare the identified ethylene biosynthesis genes between Arabidopsis (as dicotyledonous) and rice (as monocotyledonous). ResultsThe results exposed that the Arabidopsis proteins of the ethylene biosynthesis pathway had more potential glycosylation sites than rice, and 1-aminocyclopropane-1-carboxylate oxidase proteins were less phosphorylated than 1-aminocyclopropane-1-carboxylate synthase and S-adenosylmethionine proteins. According to the gene expression patterns, S-adenosylmethionine genes were more involved in the rice-ripening stage while in Arabidopsis, ACS2, and 1-aminocyclopropane-1-carboxylate oxidase genes were contributed to seed maturity. Furthermore, the result of miRNA targeting the transcript sequences showed that ath-miR843 and osa-miR1858 play a key role to regulate the post-transcription modification of S-adenosylmethionine genes in Arabidopsis and rice, respectively. The discovered cis- motifs in the promoter site of all the ethylene biosynthesis genes of A. thaliana genes were engaged to light-induced response in the cotyledon and root genes, sulfur-responsive element, dehydration, cell cycle phase-independent activation, and salicylic acid. The ACS4 protein prediction demonstrated strong protein-protein interaction in Arabidopsis, as well as, SAM2, Os04T0578000, Os01T0192900, and Os03T0727600 predicted strong protein-protein interactions in rice. ConclusionIn the current study, the complex between miRNAs with transcript sequences of ethylene biosynthesis genes in A. thaliana and O. sativa were identified, which could be helpful to understand the gene expression regulation after the transcription process. The binding sites of common transcription factors such as MYB, WRKY, and ABRE that control target genes in abiotic and biotic stresses were generally distributed in promoter sites of ethylene biosynthesis genes of A. thaliana. This was the first time to wide explore the ethylene biosynthesis pathway using bioinformatics tools that markedly showed the capability of the in silico study to integrate existing data and knowledge and furnish novel insights into the understanding of underlying ethylene biosynthesis pathway genes that will be helpful for more dissection.

Modeling-based identification of a Raptor-binding motif present in Arabidopsis ABA receptor PYL1

By employing molecular modeling of interaction simulation combined with a confirmatory yeast two-hybrid analysis, we identified the Raptor-binding region in an ABA receptor PYL1 protein of Arabidopsis. The region was a part of the C-terminal alpha-helix structure of the protein within which a phenylalanine and an aspartate in the sequence of FADTV are predicted to form critical interactions with the Raptor. Although the sequence deviates a little from the plant TOS consensus that we previously identified and defined (FSD [V/I]F) from AtS6Ks and its orthologues as well as AtATG13, the modeling data indicate that the sequence and its neighboring area are structurally capable of establishing the interaction with the Raptor in the same mode as those of other TOS motif-containing structures. This finding provides a new insight into the understanding of plant TOS motif, based upon which a putative Raptor-binding region in TAP46, another TOR substrate, is proposed.

Genome-wide identification and comprehensive analyses of NAC transcription factor gene family and expression patterns during somatic embryogenesis in Dimocarpus longan Lour

The NAM, ATAF1/2, and CUC2 form a huge plant-specific gene family of NAC TFs that are involved in the growth, development, and regulation of biotic and abiotic stress responses. Although the draft genome of longan (Dimocarpus longan Lour.) has been published, however the comprehensive data regarding the functions, evolution, and expression patterns of the NAC family are still unavailable. In this study, a comprehensive analysis of the NAC transcription factor family in longan was performed, and a total of 114 NAC genes were found. We investigated the NAC gene family exploring the phylogeny, domain conservation, intron/exon, motifs, cis-regulatory elements, protein-protein interaction, and expression profiles of RNA-seq samples in different tissues and early somatic embryogenesis of longan. Phylogenetic analysis showed that the genes with similar gene structure and motif distribution were clustered in the same group. Cis-element identification indicates the possible role of NAC genes in biological and physiological processes. Protein-protein interaction identified the DlNACs homologous with Arabidopsis proteins. We further investigated the expression pattern of DlNAC genes in different tissues (pulp, stem, large fruit, young fruit, and flower) during somatic embryogenesis at embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), and globular embryos (GE) stages. The qRT-PCR results showed that the DlNAC genes were expressed higher at EC and GE stage compared with ICpEC stage. In conclusion, our results provide insight into the evolution, diversity, and characterization of NAC genes in the longan and provide a base for understanding their biological roles and molecular mechanisms in plants.

Genome-wide investigation of proline transporter (ProT) gene family in tomato: Bioinformatics and expression analyses in response to drought stress

Proline has various functions in plants, such as growth, development and stress response to biotic and abiotic factors. Therefore, proline accumulation and transport are vital for crop production in higher quality and quantity. The present study addresses genome-wide identification and bioinformatics analyses of tomato (Solanum lycopersicum) proline transporter (ProT) genes and their expression profiles under drought stress. The analyses indicated four novel ProT genes (SlProTs) in the tomato genome and their protein lengths ranged from 439 to 452 amino acid residues. All SlProTs contained a PF01490 (transmembrane amino acid transporter protein) domain and seven exons, and they had a basic pI. The phylogeny analysis proved that monocot-dicot divergence was not present and the SlProT proteins were distinct from the ProT proteins in monocots and Arabidopsis. Based on the digital expression analysis, SlProT1 and SlProT2 genes seemed to be more active than the others in response to abiotic stress conditions. However, detected by RT-qPCR, the expression levels of all SlProT genes under drought stress were similar. The promotor analyses of SlProT genes revealed that they contained many transcription factors binding sites in cis-elements, such as MYB, Dof, Hox, bZIP, bHLH, AP2/ERF and WRKY. Finally, our findings could contribute to the understanding of SlProT genes and proline metabolism in plants.

Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of LNJ in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

Role of Arabidopsis BBX proteins in light signaling

Light regulates numerous aspects of plant growth and development like seed germination, seedling de-etiolation, pigment accumulation, cotyledon opening and flowering. Specific photoreceptors in plants sense different wavelengths of light and initiate signaling cascade leading to light-regulated responses. Between these photoreceptors and final response there are a web of transcription factors involved directly or indirectly in the signal transduction. B-Box (BBX) family of transcription factors is one of the largest family of light-regulated proteins acting as signal transducers in the light signaling pathway. B-box proteins are known for their role in light-mediated regulation of seed germination, hypocotyl elongation, flowering, circadian movements and stress tolerance. This review is an up to date compilation of the roles of BBX proteins in Arabidopsis in regulating physiological processes through red/far-red, blue and UV-B light signaling pathways in Arabidopsis. It also summarizes the roles of BBX proteins in modulating developmental pathways and stress-related signaling events in Arabidopsis and some crop species.

Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

A Poplar Rust Effector Protein Associates with Protein Disulfide Isomerase and Enhances Plant Susceptibility.

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

Humic acid enhances heat stress tolerance via transcriptional activation of Heat-Shock Proteins in Arabidopsis

Humic acid (HA) is composed of a complex supramolecular association and is produced by humification of organic matters in soil environments. HA not only improves soil fertility, but also stimulates plant growth. Although numerous bioactivities of HA have been reported, the molecular evidences have not yet been elucidated. Here, we performed transcriptomic analysis to identify the HA-prompted molecular mechanisms in Arabidopsis. Gene ontology enrichment analysis revealed that HA up-regulates diverse genes involved in the response to stress, especially to heat. Heat stress causes dramatic induction in unique gene families such as Heat-Shock Protein (HSP) coding genes including HSP101, HSP81.1, HSP26.5, HSP23.6, and HSP17.6A. HSPs mainly function as molecular chaperones to protect against thermal denaturation of substrates and facilitate refolding of denatured substrates. Interestingly, wild-type plants grown in HA were heat-tolerant compared to those grown in the absence of HA, whereas Arabidopsis HSP101 null mutant (hot1) was insensitive to HA. We also validated that HA accelerates the transcriptional expression of HSPs. Overall, these results suggest that HSP101 is a molecular target of HA promoting heat-stress tolerance in Arabidopsis. Our transcriptome information contributes to understanding the acquired genetic and agronomic traits by HA conferring tolerance to environmental stresses in plants.

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure\u2013function properties of PATELLIN2

Key messageSEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity.SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana.

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Global Quantitative Proteomics Studies Revealed Tissue-Preferential Expression and Phosphorylation of Regulatory Proteins in Arabidopsis.

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1–9 flowers, stage 10–12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

EffectorK, a comprehensive resource to mine for Ralstonia, Xanthomonas, and other published effector interactors in the Arabidopsis proteome.

Pathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant–pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large‐scale yeast two‐hybrid screenings to detect interactions between Arabidopsis thaliana proteins and effectors from two vascular bacterial pathogens: Ralstonia pseudosolanacearum and Xanthomonas campestris. We then constructed an interactomic network focused on Arabidopsis and effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein–protein interactions from 2,035 peer‐reviewed publications (48,200 Arabidopsis–Arabidopsis and 1,300 Arabidopsis–effector protein interactions). Our results show that effectors from different species interact with both common and specific Arabidopsis interactors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly “effector hubs” and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph‐oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at www.effectork.org and allows users to submit their own interactomic data.

Overproduction of plant nuclear export signals enhances diamide tolerance in Schizosaccharomyces pombe

The nuclear export signal (NES) endows a protein nuclear export ability. Surprisingly, our previous study shows that just the NES peptide of Schizosaccharomyces pombe Oxs1 (SpOxs1NES) can confer diamide tolerance by competing with transcription factor Pap1 for nuclear transport. This finding intrigued us to test the function of NESs from heterologous organisms. The Arabidopsis thaliana zinc finger transcription factor OXIDATIVE STRESS 2 (AtOXS2) is a nucleocytoplasmic shuttling protein and nearly all OXS2 members from maize and rice contain an NES. In this study, we find that the plant OXS2 members and their C-terminus (AT3 peptide) can confer diamide tolerance due to their NESs, and amino acids in non-conserved as well as conserved positions are necessary for the diamide tolerance. As in SpOxs1NES, the enhanced tolerance to diamide in fission yeast depends on Pap1. Like SpOxs1NES, OXS2 family NESs appear to compete for nuclear transport of the Pap1-like Arabidopsis protein bZIP10, as when overproduced in Arabidopsis protoplasts, bZIP10 is retained in the nucleus.

Generation of a fertile ask1 mutant uncovers a comprehensive set of SCF-mediated intracellular functions.

Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi-subunit Skp1-Cullin1-F-box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F-box proteins. Although both sequence divergence and polymorphism of F-box genes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F-box and Cullin1 in the complex, we systematically analyzed the functional influence of a well-characterized Arabidopsis Skp1-Like1 (ASK1) Ds insertion allele, ask1, in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia-0 (Col-0), we partially rescued the fertility of this otherwise sterile ask1 allele in Landsberg erecta, thus providing experimental evidence showing the polymorphic roles of SCF complexes. This ask1 mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col-0. RNA-Seq-based transcriptome analysis of ask1 uncovered a large spectrum of SCF functions, which is greater than a 10-fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far-red light/phyA-mediated photomorphogenesis. Such diverse roles are consistent with the 20-30% reduction of ubiquitylation events in ask1 estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertile ask1 mutant allowed us to uncover a comprehensive set of SCF functions.

Early Endosomal Trafficking Component BEN2/VPS45 Plays a Crucial Role in Internal Tissues in Regulating Root Growth and Meristem Size in Arabidopsis.

Polar auxin transport is involved in multiple aspects of plant development, including root growth, lateral root branching, embryogenesis, and vasculature development. PIN-FORMED (PIN) auxin efflux proteins exhibit asymmetric distribution at the plasma membrane (PM) and collectively play pivotal roles in generating local auxin accumulation, which underlies various auxin-dependent developmental processes. In previous research, it has been revealed that endosomal trafficking components BEN1/BIG5 (ARF GEF) and BEN2/VPS45 (Sec1/Munc 18 protein) function in intracellular trafficking of PIN proteins in Arabidopsis. Mutations in both BEN1 and BEN2 resulted in defects in polar PIN localization, auxin response gradients, and in root architecture. In this study, we have attempted to gain insight into the developmental roles of these trafficking components. We showed that while genetic or pharmacological disturbances of auxin distribution reduced dividing cells in the root tips and resulted in reduced root growth, the same manipulations had only moderate impact on ben1; ben2 double mutants. In addition, we established transgenic lines in which BEN2/VPS45 is expressed under control of tissue-specific promoters and demonstrated that BEN2/VPS45 regulates the intracellular traffic of PIN proteins in cell-autonomous manner, at least in stele and epidermal cells. Furthermore, BEN2/VPS45 rescued the root architecture defects when expressed in internal tissues of ben1; ben2 double mutants. These results corroborate the roles of the endosomal trafficking component BEN2/VPS45 in regulation of auxin-dependent developmental processes, and suggest that BEN2/VPS45 is required for sustainable root growth, most likely through regulation of tip-ward auxin transport through the internal tissues of root.

Ectopic expression of a poplar gene NAC13 confers enhanced tolerance to salinity stress in transgenic Nicotiana tabacum.

NACs are one of the major transcription factor families in plants which play an important role in plant growth and development, as well as in adverse stress responses. In this study, we cloned a salt-inducible NAC transcription factor gene (NAC13) from a poplar variety 84K, followed by transforming it into both Nicotiana tabacum and Arabidopsis thaliana. Stable expression analysis of 35S::NAC13-GFP fusion protein in Arabidopsis indicated that NAC13 protein was localized to the nucleus. We also obtained five transgenic tobacco lines. Evidence from morphological and physiological characterization and salt treatment analyses indicated that in the transgenic tobacco the salt tolerance was enhanced, suggesting that NAC13 gene may function as a positive regulator in tobacco responses to salt stress.

The N‐terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6-like proteins in Arabidopsis. It has been proposed that the CESA6-like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N-terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher-order structures during assembly of the CSC. We found that the mutant phenotypes of prc1-1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1-1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N-terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N-terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher-order structures in CSCs.

Proximity labeling proteomics reveals critical regulators for inner nuclear membrane protein degradation in plants

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.

Global profiling of plant nuclear membrane proteome in Arabidopsis.

The nuclear envelope (NE) is structurally and functionally vital for eukaryotic cells, yet its protein constituents and their functions are poorly understood in plants. Here, we combined subtractive proteomics and proximity-labelling technology coupled with quantitative mass spectrometry to understand the landscape of NE membrane proteins in Arabidopsis. We identified ~200 potential candidates for plant NE transmembrane (PNET) proteins, which unravelled the compositional diversity and uniqueness of the plant NE. One of the candidates, named PNET1, is a homologue of human TMEM209, a critical driver for lung cancer. A functional investigation revealed that PNET1 is a bona fide nucleoporin in plants. It displays both physical and genetic interactions with the nuclear pore complex (NPC) and is essential for embryo development and reproduction in different NPC contexts. Our study substantially enlarges the plant NE proteome and sheds new light on the membrane composition and function of the NPC.