ubiquitin-26S Proteasome Pathway Research Articles

Systematic identification of 20S proteasome substrates

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

Roles of the pepper JAZ protein CaJAZ1-03 and its interacting partner RING-type E3 ligase CaASRF1 in regulating ABA signaling and drought responses.

Plants have developed various defense mechanisms against environmental stresses by regulating hormone signaling. Jasmonic acid (JA) is a major phytohormone associated with plant defense responses. JASMONATE ZIM-DOMAIN (JAZ) proteins play a regulatory role in repressing JA signaling, impacting plant responses to both biotic and abiotic stresses. Here, we isolated 7 JAZ genes in pepper and selected CA03g31030, a Capsicum annuum JAZ1-03 (CaJAZ1-03) gene, for further study based on its expression level in response to abiotic stresses. Through virus-induced gene silencing (VIGS) in pepper and overexpression in transgenic Arabidopsis plants, we established the functional role of CaJAZ1-03. Functional studies revealed that CaJAZ1-03 dampens abscisic acid (ABA) signaling and drought stress responses. The cell-free degradation assay showed faster degradation of CaJAZ1-03 in drought- or ABA-treated pepper leaves compared to healthy leaves. Conversely, CaJAZ1-03 was completely preserved under MG132 treatment, indicating that CaJAZ1-03 stability ismodulated via the ubiquitin-26s proteasome pathway. We also found that the pepper RING-type E3 ligase CaASRF1 interacts with and ubiquitinates CaJAZ1-03. Additional cell-free degradation assays revealed a negative correlation between CaJAZ1-03 andCaASRF1 expression levels. Collectively, these findings suggest that CaJAZ1-03 negatively regulates ABA signaling and drought responses and that its protein stability is modulated by CaASRF1.

The Ubiquitin-26S Proteasome Pathway and Its Role in the Ripening of Fleshy Fruits.

The 26S proteasome is an ATP-dependent proteolytic complex in eukaryotes, which is mainly responsible for the degradation of damaged and misfolded proteins and some regulatory proteins in cells, and it is essential to maintain the balance of protein levels in the cell. The ubiquitin-26S proteasome pathway, which targets a wide range of protein substrates in plants, is an important post-translational regulatory mechanism involved in various stages of plant growth and development and in the maturation process of fleshy fruits. Fleshy fruit ripening is a complex biological process, which is the sum of a series of physiological and biochemical reactions, including the biosynthesis and signal transduction of ripening related hormones, pigment metabolism, fruit texture changes and the formation of nutritional quality. This paper reviews the structure of the 26S proteasome and the mechanism of the ubiquitin-26S proteasome pathway, and it summarizes the function of this pathway in the ripening process of fleshy fruits.

An F-box protein from wheat, TaFBA-2A, negatively regulates JA biosynthesis and confers improved salt tolerance and increased JA responsiveness to transgenic rice plants

Soil salinity is a serious problem encountered by agriculture worldwide, which will lead to many harmful effects on plant growth, development, and even crop yield. F-box protein is the core subunit of the Skp1-Cullin-F-box (SCF) complex E3 ligase and plays crucial roles in regulating the growth, development, biotic & abiotic stresses, as well as hormone signaling pathway in plants. In this study, an FBA type F-box gene TaFBA-2A was isolated from wheat (Triticum aestivum L.). This study showed that TaFBA-2A could interact with TaSKP1, and TaOPR2, the crucial enzyme involving in jasmonic acid (JA) biosynthesis. TaFBA-2A negatively regulates JA biosynthesis, probably by mediating the degradation of TaOPR2 via the ubiquitin-26S proteasome pathway. Ectopic expression of TaFBA-2A improved the salt tolerance and increased the JA responsiveness of the transgenic rice lines. In addition, some agronomic traits closely related to crop yield were significantly enhanced in the rice lines ectopic expressing TaFBA-2A. The data obtained in this study shed light on the function and mechanisms of TaFBA-2A in JA biosynthesis and the responses to salt stress and JA treatment; this study also suggested that TaFBA-2A has the potential in improving the salt tolerance and crop yield of transgenic rice plants.

Noncanonical functions of PIWIL1/piRNAs in animal male germ cells and human diseases†.

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field.

Arabidopsis F-BOX STRESS INDUCED 4 is required to repress excessive divisions in stomatal development.

During the terminal stage of stomatal development, the R2R3-MYB transcription factors FOUR LIPS (FLP/MYB124) and MYB88 limit guard mother cell division by repressing the transcript levels of multiple cell-cycle genes. In Arabidopsis thaliana possessing the weak allele flp-1, an extra guard mother cell division results in two stomata having direct contact. Here, we identified an ethylmethane sulfonate-mutagenized mutant, flp-1 xs01c, which exhibited more severe defects than flp-1 alone, producing giant tumor-like cell clusters. XS01C, encoding F-BOX STRESS-INDUCED 4 (FBS4), is preferentially expressed in epidermal stomatal precursor cells. Overexpressing FBS4 rescued the defective stomatal phenotypes of flp-1 xs01c and flp-1 mutants. The deletion or substitution of a conserved residue (Proline166) within the F-box domain of FBS4 abolished or reduced, respectively, its interaction with Arabidopsis Skp1-Like1 (ASK1), the core subunit of the Skp1/Cullin/F-box E3 ubiquitin ligase complex. Furthermore, the FBS4 protein physically interacted with CYCA2;3 and induced its degradation through the ubiquitin-26S proteasome pathway. Thus, in addition to the known transcriptional pathway, the terminal symmetric division in stomatal development is ensured at the post-translational level, such as through the ubiquitination of target proteins recognized by the stomatal lineage F-box protein FBS4.

GhUBX controlling helical growth results in production of stronger cotton fiber

SummaryCotton fiber is an excellent model for studying plant cell elongation and cell wall biogenesis as well because they are highly polarized and use conserved polarized diffuse growth mechanism. Fiber strength is an important trait among cotton fiber qualities due to ongoing changes in spinning technology. However, the molecular mechanism of fiber strength forming is obscure. Through map-based cloning, we identified the fiber strength gene GhUBX. Increasing its expression, the fiber strength of the transgenic cotton was significantly enhanced compared to the receptor W0 and the helices number of the transgenic fiber was remarkably increased. Additionally, we proved that GhUBX regulates the fiber helical growth by degrading the GhSPL1 via the ubiquitin 26S–proteasome pathway. Taken together, we revealed the internal relationship between fiber helices and fiber stronger. It will be useful for improving the fiber quality in cotton breeding and illustrating the molecular mechanism for plant twisted growth.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

F-box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis.

DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), the de novo Arabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself. Here, we conducted yeast two-hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F-BOX KELCH 1 (CFK1), as a novel DRM2-interacting partner and targets DRM2 for degradation via the ubiquitin-26S proteasome pathway in Arabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS-seq) to determine the biological significance of CFK1-mediated DRM2 degradation. Loss-of-function CFK1 leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome-wide CHH hypomethylation and transcriptional de-repression at specific DRM2 target loci. This study uncovered a distinct mechanism regulating de novo DNA methyltransferase by CFK1 to control DNA methylation level.

F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis

Aluminum (Al) toxicity is a major factor limiting crop production on acid soils, which represent over 30% of the world's arable land. Some plants have evolved mechanisms to detoxify Al. Arabidopsis, for example, secretes malate via the AtALMT1 transporter to chelate and detoxify Al. The C2H2-type transcription factor STOP1 plays a crucial role in Al resistance by inducing the expression of a set of genes, including AtALMT1 Here, we identify and characterize an F-box protein-encoding gene regulation of Atalmt1 expression 1 (RAE1) that regulates the level of STOP1. Mutation and overexpression of RAE1 increases or decreases the expression of AtALMT1 and other STOP1-regulated genes, respectively. RAE1 interacts with and promotes the degradation of STOP1 via the ubiquitin-26S proteasome pathway, while Al stress promotes the accumulation of STOP1. We find that STOP1 up-regulates RAE1 expression by directly binding to the RAE1 promoter, thus forming a negative feedback loop between STOP1 and RAE1. Our results demonstrate that RAE1 influences Al resistance through the ubiquitination and degradation of STOP1.

Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity.

Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.

GhHUB2, a ubiquitin ligase, is involved in cotton fiber development via the ubiquitin-26S proteasome pathway.

Cotton fibers, which are extremely elongated single cells of epidermal seed trichomes and have highly thickened cell walls, constitute the most important natural textile material worldwide. However, the regulation of fiber development is not well understood. Here, we report that GhHUB2, a functional homolog of AtHUB2, controls fiber elongation and secondary cell wall (SCW) deposition. GhHUB2 is ubiquitously expressed, including within fibers. Overexpression of GhHUB2 in cotton increased fiber length and SCW thickness, while RNAi knockdown of GhHUB2 resulted in shortened fibers and thinner cell walls. We found that GhHUB2 interacted with GhKNL1, a transcriptional repressor predominantly expressed in developing fibers, and that GhHUB2 ubiquitinated and degraded GhKNL1 via the ubiquitin-26S proteasome pathway. GhHUB2 negatively regulated GhKNL1 protein levels and lead to the disinhibition of genes such as GhXTH1, Gh1,3-β-G, GhCesA4, GhAGP4, GhCTL1, and GhCOBL4, thus promoting fiber elongation and enhancing SCW biosynthesis. We found that GhREV-08, a transcription factor that participates in SCW deposition and auxin signaling pathway, was a direct target of GhKNL1. In conclusion, our study uncovers a novel function of HUB2 in plants in addition to its monoubiquitination of H2B. Moreover, we provide evidence for control of the fiber development by the ubiquitin-26S proteasome pathway.

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway.

Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.

Many flowering plants show self-incompatibility, an intra-specific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote out-crossing. In Petunia, the polymorphic S-locus determines self/non-self recognition. The locus contains a gene encoding an S-RNase, which controls pistil specificity, and multiple S-locus F-box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F-box) complex that is responsible for mediating degradation of non-self S-RNase(s), with which the SLF interacts, via the ubiquitin-26S proteasome pathway. A complete set of SLFs is required to detoxify all non-self S-RNases to allow cross-compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin-26S proteasome pathway, and identify an 18 amino acid sequence in the C-terminal region of S2 -SLF1 (SLF1 of S2 haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S2 haplotype and S3 haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S2 -SLF1 stabilized the protein but abolished its function in self-incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self-incompatibility.

The non-catalytic N-terminal domain of ACS7 is involved in the post-translational regulation of this gene in Arabidopsis.

Post-transcriptional control of the expression of the 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family is important for maintaining appropriate levels of ethylene production. However, the molecular mechanism underlying the post-transcriptional regulation of type 3 ACS proteins remains unclear. Multiple sequence alignment revealed that the N-terminus of type 3 ACSs was longer than that of other ACSs. Fusing the N-terminal 54 residues of ACS7, the sole type 3 ACS in Arabidopsis, to the β-glucuronidase (GUS) reporter significantly decreased the stability of N(7(1-54))-GUS fusion protein. Among these 54 residues, residues 1-14 conferred this negative effect on the GUS fusion gene. Consistently, a truncated form of ACS7 lacking residues 1-14 was more stable than full-length ACS7 when transgenically expressed in Arabidopsis and led to a more severe ethylene response phenotype in the light-grown transgenic seedlings. Interestingly, the ACS7 N-terminus had no effect on the stability of N(7)-GUS and ACS7 proteins at the etiolated seedling stage. Both exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) treatment and salt stress could rescue the levels of accumulation of N(7)-GUS fusion protein in light-grown seedlings. These results suggest that the non-catalytic N-terminus of ACS7 is involved in its own post-translational regulation. The proteasome inhibitor MG132 suppressed degradation of full-length ACS7 in vivo, but had little effect on the N-terminal truncated form of ACS7, indicating that the N-terminus mediates the regulation of ACS7 stability through the ubiquitin-26S proteasome pathway.

The Progress on the Ubiquitin /26S Proteasome Pathway in Plants

Ubiquitin /26S proteasome pathway( UPP) is the most effective,highly selective proteolytic pathway. It mediates 80% to 85% of the protein degradation in eukaryotic cell,participates in various cellular life courses,and plays an important role in maintaining the normal physiological function of the cells. The results show that many aspects of plant growth and development as well as some processes such as drought stress response are all affected by this pathway. It summarizes ubiquitin /26S proteasome pathway,which roles in the processes of plant growth and development,and emphatically elaborates the progress of the plant drought stress response and its mechanism mediated by the ubiquitin-protein ligase E3.

Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock

To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes – ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) – of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis—wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1–1μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.

Rice APC/CTE controls tillering by mediating the degradation of MONOCULM 1

Rice MONOCULM 1 (MOC1) and its orthologues LS/LAS (lateral suppressor in tomato and Arabidopsis) are key promoting factors of shoot branching and tillering in higher plants. However, the molecular mechanisms regulating MOC1/LS/LAS have remained elusive. Here we show that the rice tiller enhancer (te) mutant displays a drastically increased tiller number. We demonstrate that TE encodes a rice homologue of Cdh1, and that TE acts as an activator of the anaphase promoting complex/cyclosome (APC/C) complex. We show that TE coexpresses with MOC1 in the axil of leaves, where the APC/CTE complex mediates the degradation of MOC1 by the ubiquitin–26S proteasome pathway, and consequently downregulates the expression of the meristem identity gene Oryza sativa homeobox 1, thus repressing axillary meristem initiation and formation. We conclude that besides having a conserved role in regulating cell cycle, APC/CTE has a unique function in regulating the plant-specific postembryonic shoot branching and tillering, which are major determinants of plant architecture and grain yield.

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in Cyclamen persicum

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.