Salicylic acid attenuates brassinosteroid signaling via protein de-S-acylation.

Brassinosteroids (BRs) are important plant hormones involved in many aspects of development. Here, we show that BRASSINOSTEROID SIGNALING KINASEs (BSKs), key components of the BR pathway, are precisely controlled via de-S-acylation mediated by the defense hormone salicylic acid (SA). Most Arabidopsis BSK members are substrates of S-acylation, a reversible protein lipidation that is essential for their membrane localization and physiological function. We establish that SA interferes with the plasma membrane localization and function of BSKs by decreasing their S-acylation levels, identifying ABAPT11 (ALPHA/BETA HYDROLASE DOMAIN-CONTAINING PROTEIN 17-LIKE ACYL PROTEIN THIOESTERASE 11) as an enzyme whose expression is quickly induced by SA. ABAPT11 de-S-acylates most BSK family members, thus integrating BR and SA signaling for the control of plant development. In summary, we show that BSK-mediated BR signaling is regulated by SA-induced protein de-S-acylation, which improves our understanding of the function of protein modifications in plant hormone cross talk.

Heat-induced SUMOylation differentially affects bacterial effectors in plant cells.

Bacterial pathogens deliver effectors into host cells to suppress immunity. How host cells target these effectors is critical in pathogen-host interactions. SUMOylation, an important type of posttranslational modification in eukaryotic cells, plays a critical role in immunity, but its effect on bacterial effectors remains unclear in plant cells. In this study, using bioinformatic and biochemical approaches, we found that at least 16 effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 are SUMOylated by the enzyme cascade from Arabidopsis thaliana. Mutation of SUMOylation sites on the effector HopB1 enhances its function in the induction of plant cell death via stability attenuation of a plant receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1. By contrast, SUMOylation is essential for the function of another effector, HopG1, in the inhibition of mitochondria activity and jasmonic acid signaling. SUMOylation of both HopB1 and HopG1 is increased by heat treatment, and this modification modulates the functions of these 2 effectors in different ways in the regulation of plant survival rates, gene expression, and bacterial infection under high temperatures. Therefore, the current work on the SUMOylation of effectors in plant cells improves our understanding of the function of dynamic protein modifications in plant-pathogen interactions in response to environmental conditions.

S-acylation of a non-secreted peptide controls plant immunity via secreted-peptide signal activation.

Small peptides modulate multiple processes in plant cells, but their regulation by post-translational modification remains unclear. ROT4 (ROTUNDIFOLIA4) belongs to a family of Arabidopsis non-secreted small peptides, but knowledge on its molecular function and how it is regulated is limited. Here, we find that ROT4 is S-acylated in plant cells. S-acylation is an important form of protein lipidation, yet so far it has not been reported to regulate small peptides in plants. We show that this modification is essential for the plasma membrane association of ROT4. Overexpression of S-acylated ROT4 results in a dramatic increase in immune gene expression. S-acylation of ROT4 enhances its interaction with BSK5 (BRASSINOSTEROID-SIGNALING KINASE 5) to block the association between BSK5 and PEPR1 (PEP RECEPTOR1), a receptor kinase for secreted plant elicitor peptides (PEPs), thereby activating immune signaling. Phenotype analysis indicates that S-acylation is necessary for ROT4 functions in pathogen resistance, PEP response, and the regulation of development. Collectively, our work reveals an important role for S-acylation in the cross-talk of non-secreted and secreted peptide signaling in plant immunity.

A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair.

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.

The chromosome-scale assembly of the Salvia rosmarinus genome provides insight into carnosic acid biosynthesis.

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.

SUMO E3 ligase AtMMS21-dependent SUMOylation of AUXIN/INDOLE-3-ACETIC ACID 17 regulates auxin signaling.

Changes in plant auxin levels can be perceived and converted into cellular responses by auxin signal transduction. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are auxin transcriptional inhibitors that play important roles in regulating auxin signal transduction. The stability of Aux/IAA proteins is important for transcription initiation and downstream auxin-related gene expression. Here, we report that the Aux/IAA protein IAA17 interacts with the small ubiquitin-related modifier (SUMO) E3 ligase METHYL METHANESULFONATE-SENSITIVE 21 (AtMMS21) in Arabidopsis (Arabidopsis thaliana). AtMMS21 regulated the SUMOylation of IAA17 at the K41 site. Notably, root length was suppressed in plants overexpressing IAA17, whereas the roots of K41-mutated IAA17 transgenic plants were not significantly different from wild-type roots. Biochemical data indicated that K41-mutated IAA17 or IAA17 in the AtMMS21 knockout mutant was more likely to be degraded compared with nonmutated IAA17 in wild-type plants. In conclusion, our data revealed a role for SUMOylation in the maintenance of IAA17 protein stability, which contributes to improving our understanding of the mechanisms of auxin signaling.

An ABHD17-like hydrolase screening system to identify de-S-acylation enzymes of protein substrates in plant cells.

Protein S-acylation is an important post-translational modification in eukaryotes, regulating the subcellular localization, trafficking, stability, and activity of substrate proteins. The dynamic regulation of this reversible modification is mediated inversely by protein S-acyltransferases and de-S-acylation enzymes, but the de-S-acylation mechanism remains unclear in plant cells. Here, we characterized a group of putative protein de-S-acylation enzymes in Arabidopsis thaliana, including 11 members of Alpha/Beta Hydrolase Domain-containing Protein 17-like acyl protein thioesterases (ABAPTs). A robust system was then established for the screening of de-S-acylation enzymes of protein substrates in plant cells, based on the effects of substrate localization and confirmed via the protein S-acylation levels. Using this system, the ABAPTs, which specifically reduced the S-acylation levels and disrupted the plasma membrane localization of five immunity-related proteins, were identified respectively in Arabidopsis. Further results indicated that the de-S-acylation of RPM1-Interacting Protein 4, which was mediated by ABAPT8, resulted in an increase of cell death in Arabidopsis and Nicotiana benthamiana, supporting the physiological role of the ABAPTs in plants. Collectively, our current work provides a powerful and reliable system to identify the pairs of plant protein substrates and de-S-acylation enzymes for further studies on the dynamic regulation of plant protein S-acylation.

SUMOylation facilitates the assembly of a Nuclear Factor-Y complex to enhance thermotolerance in Arabidopsis.

Heat stress (HS) has serious negative effects on plant development and has become a major threat to agriculture. A rapid transcriptional regulatory cascade has evolved in plants in response to HS. Nuclear Factor-Y (NF-Y) complexes are critical for this mechanism, but how NF-Y complexes are regulated remains unclear. In this study, we identified NF-YC10 (NF-Y subunit C10), a central regulator of the HS response in Arabidopsis thaliana, as a substrate of SUMOylation, an important post-translational modification. Biochemical analysis showed that the SUMO ligase SIZ1 (SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1) interacts with NF-YC10 and enhances its SUMOylation during HS. The SUMOylation of NF-YC10 facilitates its interaction with and the nuclear translocation of NF-YB3, in which the SUMO interaction motif (SIM) is essential for its efficient association with NF-YC10. Further functional analysis indicated that the SUMOylation of NF-YC10 and the SIM of NF-YB3 are critical for HS-responsive gene expression and plant thermotolerance. These findings uncover a role for the SIZ1-mediated SUMOylation of NF-YC10 in NF-Y complex assembly under HS, providing new insights into the role of a post-translational modification in regulating transcription during abiotic stress responses in plants.

Importation of chloroplast proteins under heat stress is facilitated by their SUMO conjugations.

Chloroplasts are hypersensitive to heat stress (HS). SUMOylation, a critical post-translational modification, is conservatively involved in HS responses. However, the functional connection between SUMOylation and chloroplasts under HS remains to be studied. The bioinformatics, biochemistry, and cell biology analyses were used to detect the SUMOylation statuses of Arabidopsis nuclear-encoded chloroplast proteins and the effect of SUMOylation on subcellular localization of these proteins under HS. PSBR, a subunit of photosystem II, was used as an example for a detailed investigation of functional mechanisms. After a global SUMOylation site prediction of nuclear-encoded chloroplast proteins, biochemical data showed that most of the selected candidates are modified by SUMO3 in the cytoplasm. The chloroplast localization of these SUMOylation targets under long-term HS is partially maintained by the SUMO ligase AtSIZ1. The HS-induced SUMOylation on PSBR contributes to the maintenance of its chloroplast localization, which is dependent on its chloroplast importation efficiency correlated to phosphorylation. The complementation analysis provided evidence that SUMOylation is essential for the physiological function of PSBR under HS. Our study illustrated a general regulatory mechanism of SUMOylation in maintaining the chloroplast protein importation during HS and provided hints for further investigation on protein modifications associated with plant organelles under stress conditions.

A SWI/SNF subunit regulates chromosomal dissociation of structural maintenance complex 5 during DNA repair in plant cells.

DNA damage decreases genome stability and alters genetic information in all organisms. Conserved protein complexes have been evolved for DNA repair in eukaryotes, such as the structural maintenance complex 5/6 (SMC5/6), a chromosomal ATPase involved in DNA double-strand break (DSB) repair. Several factors have been identified for recruitment of SMC5/6 to DSBs, but this complex is also associated with chromosomes under normal conditions; how SMC5/6 dissociates from its original location and moves to DSB sites is completely unknown. In this study, we determined that SWI3B, a subunit of the SWI/SNF complex, is an SMC5-interacting protein in Arabidopsis thialiana Knockdown of SWI3B or SMC5 results in increased DNA damage accumulation. During DNA damage, SWI3B expression is induced, but the SWI3B protein is not localized at DSBs. Notably, either knockdown or overexpression of SWI3B disrupts the DSB recruitment of SMC5 in response to DNA damage. Overexpression of a cotranscriptional activator ADA2b rescues the DSB localization of SMC5 dramatically in the SWI3B-overexpressing cells but only weakly in the SWI3B knockdown cells. Biochemical data confirmed that ADA2b attenuates the interaction between SWI3B and SMC5 and that SWI3B promotes the dissociation of SMC5 from chromosomes. In addition, overexpression of SMC5 reduces DNA damage accumulation in the SWI3B knockdown plants. Collectively, these results indicate that the presence of an appropriate level of SWI3B enhances dissociation of SMC5 from chromosomes for its further recruitment at DSBs during DNA damage in plant cells.

SUMOylation Stabilizes the Transcription Factor DREB2A to Improve Plant Thermotolerance.

Heat stress (HS) has serious effects on plant development, resulting in heavy agricultural losses. A critical transcription factor network is involved in plant adaptation to high temperature. DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) is a key transcription factor that functions in plant thermotolerance. The DREB2A protein is unstable under normal temperature and is degraded by the 26S proteasome; however, the mechanism by which DREB2A protein stability dramatically increases in response to HS remains poorly understood. In this study, we found that the DREB2A protein of Arabidopsis (Arabidopsis thaliana) is stabilized under high temperature by the posttranslational modification SUMOylation. Biochemical data indicated that DREB2A is SUMOylated at K163, a conserved residue adjacent to the negative regulatory domain during HS. SUMOylation of DREB2A suppresses its interaction with BPM2, a ubiquitin ligase component, consequently increasing DREB2A protein stability under high temperature. In addition, analysis of plant heat tolerance and marker gene expression indicated that DREB2A SUMOylation is essential for its function in the HS response. Collectively, our data reveal a role for SUMOylation in the maintenance of DREB2A stability under high temperature, thus improving our understanding of the regulatory mechanisms underlying HS response in plant cells.

Protein modification: A critical modulator in the interaction between geminiviruses and host plants.

Geminiviruses are a large group of single-stranded DNA viruses that infect plants and cause severe agricultural losses worldwide. Given geminiviruses only have small genomes that encode a few proteins, viral factors have to interact with host components to establish an environment suitable for virus infection, whilst the host immunity system recognizes and targets these viral components during infection. Post-translational protein modifications, such as phosphorylation, lipidation, ubiquitination, SUMOylation, acetylation and methylation, have been reported to be critical during the interplay between host plants and geminiviruses. Here we summarize the research progress, including phosphorylation and lipidation which usually control the activity and localization of viral factors; as well as ubiquitination and histone modification which are predominantly interfered with by viral components. We also discuss the dynamic competition on protein modifications between host defence and geminivirus efficient infection, as well as potential applications of protein modifications in geminivirus resistance. The summary and perspective of this topic will improve our understanding on the mechanism of geminivirus-plant interaction and contribute to further protection of plants from virus infection.

Danger peptide signaling enhances internalization of a geminivirus symptom determinant in plant cells during infection.

Geminiviruses are DNA viruses that cause severe diseases in diverse species of plants, resulting in considerable agricultural losses worldwide. C4 proteins are a major symptom determinant in several geminiviruses, including Beet severe curly top virus (BSCTV). Here, we uncovered a novel mechanism by which danger peptide signaling enhances the internalization of BSCTV C4 in plant cells. Previous studies showed that this signaling is important for activation of bacterium- and fungus-triggered immune responses, but its function in plant-virus interactions was previously unknown. Pep1 RECEPTOR1 (PEPR1) and PEPR2 are receptor kinases recognized by Peps (plant elicitor peptides) in the danger peptide pathway. We found that BSCTV C4 up-regulated and interacted with PEPR2 but not PEPR1. The Pep1-PEPR2 complex stimulated the internalization of C4 in both Arabidopsis and Nicotiana benthamiana cells. Furthermore, C4 induced callus formation in Arabidopsis, which was suppressed by PEPR2 overexpression but enhanced in the pepr2 mutants. In the presence of Pep1, overexpression of PEPR2 suppressed BSCTV infection in N. benthamiana. Exogenous Pep1 also reduced BSCTV infection in Arabidopsis in a PEPR2-dependent manner. Thus, PEPR2 recognizes the symptom determinant C4 and enhances its internalization mediated by danger peptides, suppressing BSCTV infection.

The Transcriptional Coactivator ADA2b Recruits a Structural Maintenance Protein to Double-Strand Breaks during DNA Repair in Plants.

DNA damage occurs in all cells and can hinder chromosome stability and cell viability. Structural Maintenance of Chromosomes5/6 (SMC5/6) is a protein complex that functions as an evolutionarily conserved chromosomal ATPase critical for repairing DNA double-strand breaks (DSBs). However, the mechanisms regulating this complex in plants are poorly understood. Here, we identified the transcriptional coactivator ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b) as an interactor of SMC5 in Arabidopsis (Arabidopsis thaliana). ADA2b is a conserved component of the Spt-Ada-Gcn5 acetyltransferase complex, which functions in transcriptional regulation. Characterization of mutant and knockdown Arabidopsis lines showed that disruption of either SMC5 or ADA2b resulted in enhanced DNA damage. Both SMC5 and ADA2b were associated with γ-H2AX, a marker of DSBs, and the recruitment of SMC5 onto DSBs was dependent on ADA2b. In addition, overexpression of SMC5 in the ada2b mutant background stimulated cell death. Collectively, our results show that the interaction between ADA2b and SMC5 mediates DNA repair in plant cells, suggesting a functional association between these conserved proteins and further elucidating mechanisms of DNA damage repair in plants.

The Arabidopsis SUMO E3 Ligase AtMMS21 Dissociates the E2Fa/DPa Complex in Cell Cycle Regulation.

Development requires the proper execution and regulation of the cell cycle via precise, conserved mechanisms. Critically, the E2F/DP complex controls the expression of essential genes during cell cycle transitions. Here, we discovered the molecular function of the Arabidopsis thaliana SUMO E3 ligase METHYL METHANESULFONATE SENSITIVITY GENE21 (AtMMS21) in regulating the cell cycle via the E2Fa/DPa pathway. DPa was identified as an AtMMS21-interacting protein and AtMMS21 competes with E2Fa for interaction with DPa. Moreover, DPa is a substrate for SUMOylation mediated by AtMMS21, and this SUMOylation enhances the dissociation of the E2Fa/DPa complex. AtMMS21 also affects the subcellular localization of E2Fa/DPa. The E2Fa/DPa target genes are upregulated in the root of mms21-1 and mms21-1 mutants showed increased endoreplication. Overexpression of DPa affected the root development of mms21-1, and overexpression of AtMMS21 completely recovered the abnormal phenotypes of 35S:E2Fa-DPa plants. Our results suggest that AtMMS21 dissociates the E2Fa/DPa complex via competition and SUMOylation in the regulation of plant cell cycle.

A SUMO Ligase AtMMS21 Regulates the Stability of the Chromatin Remodeler BRAHMA in Root Development.

Chromatin remodeling is essential for gene expression regulation in plant development and response to stresses. Brahma (BRM) is a conserved ATPase in the SWI/SNF chromatin remodeling complex and is involved in various biological processes in plant cells, but the regulation mechanism on BRM protein remains unclear. Here, we report that BRM interacts with AtMMS21, a SUMO ligase in Arabidopsis (Arabidopsis thaliana). The interaction was confirmed in different approaches in vivo and in vitro. The mutants of BRM and AtMMS21 displayed a similar defect in root development. In the mms21-1 mutant, the protein level of BRM-GFP was significantly lower than that in wild type, but the RNA level of BRM did not change. Biochemical evidence indicated that BRM was modified by SUMO3, and the reaction was enhanced by AtMMS21. Furthermore, overexpression of wild-type AtMMS21 but not the mutated AtMMS21 without SUMO ligase activity was able to recover the stability of BRM in mms21-1 Overexpression of BRM in mms21-1 partially rescued the developmental defect of roots. Taken together, these results supported that AtMMS21 regulates the protein stability of BRM in root development.

S-acylation of a geminivirus C4 protein is essential for regulating the CLAVATA pathway in symptom determination.

Geminiviruses, such as beet severe curly top virus (BSCTV), are a group of DNA viruses that cause severe plant diseases and agricultural losses. The C4 protein is a major symptom determinant in several geminiviruses; however, its regulatory mechanism and molecular function in plant cells remain unclear. Here, we show that BSCTV C4 is S-acylated in planta, and that this post-translational lipid modification is necessary for its membrane localization and functions, especially its regulation of shoot development of host plants. Furthermore, the S-acylated form of C4 interacts with CLAVATA 1 (CLV1), an important receptor kinase in meristem maintenance, and consequentially affects the expression of WUSCHEL, a major target of CLV1. The abnormal development of siliques in Arabidopsis thaliana infected with BSCTV is also dependent on the S-acylation of C4, implying a potential role of CLAVATA signaling in this process. Collectively, our results show that S-acylation is essential for BSCTV C4 function, including the regulation of the CLAVATA pathway, during geminivirus infection.

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

BACKGROUND: SUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium. RESULTS: In Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1. CONCLUSIONS: Our results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.

OsAGSW1, an ABC1-like kinase gene, is involved in the regulation of grain size and weight in rice.

Grain shape and weight are two determining agronomic traits of rice yield. ABC1 (Activity of bc1 complex) is a newly found atypical kinase in plants. Here, we report on an ABC1 protein kinase gene, OsAGSW1 (ABC1-like kinase related to Grain size and Weight). Expression of OsAGSW1-GFP in rice revealed that OsAGSW1 is localized to the chloroplasts in rice. Analysis of OsAGSW1 promoter::ß-glucuronidase transgenic rice indicated that this gene was highly expressed in vascular bundles in shoot, hull and caryopsis. Furthermore, OsAGSW1-RNAi and overexpressed transgenic rice lines were generated. Stable transgenic lines overexpressing OsAGSW1 exhibited a phenotype with a significant increase in grain size, grain weight, grain filling rate and 1000-grain weight compared with the wild-type and RNAi transgenic plants. Microscopy analysis showed that spikelet hulls just before heading were different in the OsAGSW1-overexpressed plants compared with wild-type and OsAGSW1 RNAi rice. Further cytological analysis showed that the number of external parenchyma cells in rice hulls of OsAGSW1-overexpressed plants increased, leading to wider and longer spikelet hulls than those of the wild-type and OsAGSW1-RNAi plants. The vascular cross-sectional area in lemma, carpopodium and ovules also strikingly increased and area of both xylem and phloem were enlarged in the OsAGSW1-overexpressed plants. Thus, our results demonstrated that OsAGSW1 plays an important role in seed shape and size of rice by regulating the number of external parenchyma cells and the development of vascular bundles, providing a new insight into the functions of ABC1 genes in plants.

Two homologous protein S-acyltransferases, PAT13 and PAT14, cooperatively regulate leaf senescence in Arabidopsis.

Lipid modification on the cysteine residues of proteins, known as S-palmitoylation or S-acylation, regulates the subcellular localization and the function of proteins. S-acylation is catalysed by a group of protein acyltransferases (PATs) with a conserved Asp-His-His-Cys (DHHC) motif. The molecular function of S-acylation has been studied in details in yeast and mammalian cells, but its role in plant cells remains unclear. Here it is reported that the expression of two homologous protein acyltransferases- PAT13 and PAT14 -was moderately increased in the older leaves of Arabidopsis. The double mutant of PAT13 and PAT14 displayed a severely early leaf senescence phenotype. The phenotype was complemented by PAT13 or PAT14 overexpression in the double mutant, confirming the roles of PAT13 and PAT14 in this process. Furthermore, the levels of reactive oxygen species (ROS) and cell death were dramatically induced in the double mutant. To investigate the molecular functions of PAT13 and PAT14, their potential S-acylation substrates were predicted by bioinformatics methods. The subcellular localization and S-acylation of a candidate substrate NITRIC OXIDE ASSOCIATED 1 (NOA1), which also plays a role in leaf senescence control, were partially disrupted in the protoplasts of the double mutant. Impairment of S-acylation on NOA1 affected its subcellular localization and its function in leaf senescence regulation. Conclusively, protein S-acyltransferases PAT13 and PAT14 are involved in leaf senescence control- possibly via NOA1 S-acylation-, providing a new sight into the regulation mechanism of S-acylation in leaf senescence.