Comparative gene retention analysis in barley, wild emmer, and bread wheat pangenome lines reveals factors affecting gene retention following gene duplication.

BACKGROUND: Gene duplication is a prevalent phenomenon and a major driving force underlying genome evolution. The process leading to the fixation of gene duplicates following duplication is critical to understand how genome evolves but remains fragmentally understood. Most previous studies on gene retention are based on gene duplicate analyses in single reference genome. No population-based comparative gene retention analysis has been performed to date. RESULTS: Taking advantage of recently published genomic data in Triticeae, we dissected a divergent homogentisate phytyltransferase (HPT2) lineage caught in the middle stage of gene fixation following duplication. The presence/absence of HPT2 in barley (diploid), wild emmer (tetraploid), and bread wheat (hexaploid) pangenome lines appears to be associated with gene dosage constraint and environmental adaption. Based on these observations, we adopted a phylogeny-based orthology inference approach and performed comparative gene retention analyses across barley, wild emmer, and bread wheat. This led to the identification of 326 HPT2-pattern-like genes at whole genome scale, representing a pool of gene duplicates in the middle stage of gene fixation. Majority of these HPT2-pattern-like genes were identified as small-scale duplicates, such as dispersed, tandem, and proximal duplications. Natural selection analyses showed that HPT2-pattern-like genes have experienced relaxed selection pressure, which is generally accompanied with partial positive selection and transcriptional divergence. Functional enrichment analyses showed that HPT2-pattern-like genes are over-represented with molecular-binding and defense response functions, supporting the potential role of environmental adaption during gene retention. We also observed that gene duplicates from larger gene family are more likely to be lost, implying a gene dosage constraint effect. Further comparative gene retention analysis in barley and bread wheat pangenome lines revealed combined effects of species-specific selection and gene dosage constraint. CONCLUSIONS: Comparative gene retention analyses at the population level support gene dosage constraint, environmental adaption, and species-specific selection as three factors that may affect gene retention following gene duplication. Our findings shed light on the evolutionary process leading to the retention of newly formed gene duplicates and will greatly improve our understanding on genome evolution via duplication.

Identification of New ATG8s-Binding Proteins with Canonical LC3-Interacting Region in Autophagosomes of Barley Callus.

Autophagy is essential to maintain cellular homeostasis for normal cell growth and development. In selective autophagy, ATG8 plays a crucial role in cargo target recognition by binding to various adaptors and receptors with the ATG8-interacting motif, also known as the LC3-interacting region (LIR). However, the process of autophagy in the callus, as a proliferating cell type, is largely unknown. In this study, we overexpressed green fluorescent protein (GFP)-ATG8a and GFP-ATG8b transgenic barley callus and checked their autophagic activities. We identified five new ATG8 candidate interactors containing the canonical LIR motif by using immunoprecipitation coupled with mass spectrometry: RPP3, COPE, NCLN, RAE1, and CTSL. The binding activities between these candidate interactors and ATG8 were further demonstrated in the punctate structure. Notably, RPP3 was colocalized in ATG8-labeled autophagosomes under tunicamycin-induced ER stress. GST pull-down assays showed that the interaction between RPP3 and ATG8 could be prevented by mutating the LIRs region of RPP3 or the LIR docking site (LDS) of ATG8, suggesting that RPP3 directly interacted with ATG8 in an LIR-dependent manner via the LDS. Our findings would provide the basis for further investigations on novel receptors and functions of autophagy in plants, especially in the physiological state of cell de-differentiation.

Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis.

BACKGROUND: Barley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive. RESULTS: This study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity. CONCLUSIONS: We identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Functional analysis of auxin receptor OsTIR1/OsAFB family members in rice grain yield, tillering, plant height, root system, germination, and auxinic herbicide resistance.

Auxin regulates almost every aspect of plant growth and development and is perceived by the TIR1/AFB auxin co-receptor proteins differentially acting in concert with specific Aux/IAA transcriptional repressors. Little is known about the diverse functions of TIR1/AFB family members in species other than Arabidopsis. We created targeted OsTIR1 and OsAFB2-5 mutations in rice using CRISPR/Cas9 genome editing, and functionally characterized the roles of these five members in plant growth and development and auxinic herbicide resistance. Our results demonstrated that functions of OsTIR1/AFB family members are partially redundant in grain yield, tillering, plant height, root system and germination. Ostir1, Osafb2 and Osafb4 mutants exhibited more severe phenotypes than Osafb3 and Osafb5. The Ostir1Osafb2 double mutant displays extremely severe defects in plant development. All five OsTIR1/AFB members interacted with OsIAA1 and OsIAA11 proteins in vivo. Root elongation assay showed that each Ostir1/afb2-5 mutant was resistant to 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. Notably, only the Osafb4 mutants were strongly resistant to the herbicide picloram, suggesting that OsAFB4 is a unique auxin receptor in rice. Our findings demonstrate similarities and specificities of auxin receptor TIR1/AFB proteins in rice, and could offer the opportunity to modify effective herbicide-resistant alleles in agronomically important crops.

The SAUR41 subfamily of SMALL AUXIN UP RNA genes is abscisic acid inducible to modulate cell expansion and salt tolerance in Arabidopsis thaliana seedlings.

BACKGROUND AND AIMS: Most primary auxin response genes are classified into three families: AUX/IAA, GH3 and SAUR genes. Few studies have been conducted on Arabidopsis thaliana SAUR genes, possibly due to genetic redundancy among different subfamily members. Data mining on arabidopsis transcriptional profiles indicates that the SAUR41 subfamily members of SMALL AUXIN UP RNA genes are, strikingly, induced by an inhibitory phytohormone, abscisic acid (ABA). We aimed to reveal the physiological roles of arabidopsis SAUR41 subfamily genes containing SAUR40, SAUR41, SAUR71 and SAUR72. METHODS: Transcriptional responses of arabidopsis SAUR41 genes to phytohormones were determined by quantitative real-time PCR. Knock out of SAUR41 genes was carried out with the CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing technique. The saur41/40/71/72 quadruple mutants, SAUR41 overexpression lines and the wild type were subjected to ultrastructural observation, transcriptome analysis and physiological characterization. KEY RESULTS: Transcription of arabidopsis SAUR41 subfamily genes is activated by ABA but not by gibberellic acids and brassinosteroids. Quadruple mutations in saur41/40/71/72 led to reduced cell expansion/elongation in cotyledons and hypocotyls, opposite to the overexpression of SAUR41; however, an irregular arrangement of cell size and shape was observed in both cases. The quadruple mutants had increased transcription of calcium homeostasis/signalling genes in seedling shoots, and the SAUR41 overexpression lines had decreased transcription of iron homeostasis genes in roots and increased ABA biosynthesis in shoots. Notably, both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress during seedling establishment, whereas specific expression of SAUR41 under the ABA-responsive RD29A (Responsive to Desiccation 29A) promoter in the quadruple mutants rescued the inhibitory effect of salt stress. CONCLUSIONS: The SAUR41 subfamily genes of arabidopsis are ABA inducible to modulate cell expansion, ion homeostasis and salt tolerance. Our work may provide new candidate genes for improvement of plant abiotic stress tolerance.

Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing.

BACKGROUND AND AIMS: Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. METHODS: Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. KEY RESULTS: Mutagenesis efficiency among T0 regenerated plantlets was 50-65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. CONCLUSIONS: Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.

Epigenetic regulation of miR396 expression by SWR1-C and the effect of miR396 on leaf growth and developmental phase transition in Arabidopsis.

In this study, we investigated the regulatory function of miR396 in the phase transition in Arabidopsis thaliana. Using AtMIR396a/b knockout mutants generated through clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-directed genome editing, we showed that miR396 negatively regulates the leaf size and vegetative phase transition, and the first leaf with abaxial trichomes appeared earlier in the mir396ab double mutant than in the wild type (WT) and was significantly delayed in miR396 overexpression lines. Moreover, mir396ab exhibited early flowering, whereas 35S:MIR396a/b and cib4-1 delayed flowering, and the flowering time was negatively correlated with FT gene expression. Furthermore, in arp6 and pie1 mutants, which are deficient in the ATP-dependent chromatin remodeling complex (SWR1-C), miR396 expression was significantly repressed. Compared with the WT, reduced H2A.Z deposit and stronger relative nucleosome occupancy in the promoter region of MIR396a was found in the arp6 mutant, indicating that SWR1-C contributes to the transcriptional activation of MIR396a via nucleosome dynamics. In addition, miR396 displayed specific spatio-temporal expression patterns in the leaf, which was altered in arp6 and pie1, and therefore affected the transcript levels of CIB4 and FT in these mutants. We propose that miR396 is not only a marker of cell differentiation, but also an age signal for leaf development and phase change. Meanwhile, SWR1-C-mediated epigenetic regulation contributes to the age-dependent enhancement of miR396 expression and differential miR396 accumulation among leaves.

Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites.

The primary cilium is a microtubule-based organelle that functions in sensory and signalling pathways. Defects in ciliogenesis can lead to a group of genetic syndromes known as ciliopathies. However, the regulatory mechanisms of primary ciliogenesis in normal and cancer cells are incompletely understood. Here we demonstrate that autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles and protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction. We show that the population of OFD1 at the centriolar satellites is rapidly degraded by autophagy upon serum starvation. In autophagy-deficient Atg5 or Atg3 null mouse embryonic fibroblasts, OFD1 accumulates at centriolar satellites, leading to fewer and shorter primary cilia and a defective recruitment of BBS4 (Bardet-Biedl syndrome 4) to cilia. These defects are fully rescued by OFD1 partial knockdown that reduces the population of OFD1 at centriolar satellites. More strikingly, OFD1 depletion at centriolar satellites promotes cilia formation in both cycling cells and transformed breast cancer MCF7 cells that normally do not form cilia. This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis.

Cistrome Data Browser: a data portal for ChIP-Seq and chromatin accessibility data in human and mouse.

Chromatin immunoprecipitation, DNase I hypersensitivity and transposase-accessibility assays combined with high-throughput sequencing enable the genome-wide study of chromatin dynamics, transcription factor binding and gene regulation. Although rapidly accumulating publicly available ChIP-seq, DNase-seq and ATAC-seq data are a valuable resource for the systematic investigation of gene regulation processes, a lack of standardized curation, quality control and analysis procedures have hindered extensive reuse of these data. To overcome this challenge, we built the Cistrome database, a collection of ChIP-seq and chromatin accessibility data (DNase-seq and ATAC-seq) published before January 1, 2016, including 13 366 human and 9953 mouse samples. All the data have been carefully curated and processed with a streamlined analysis pipeline and evaluated with comprehensive quality control metrics. We have also created a user-friendly web server for data query, exploration and visualization. The resulting Cistrome DB (Cistrome Data Browser), available online at http://cistrome.org/db, is expected to become a valuable resource for transcriptional and epigenetic regulation studies.

miR393-Mediated Auxin Signaling Regulation is Involved in Root Elongation Inhibition in Response to Toxic Aluminum Stress in Barley.

High-throughput small RNA sequencing has identified several potential aluminum (Al)-responsive microRNAs (miRNAs); however, their regulatory role remains unknown. Here, we identified two miR393 family members in barley, and confirmed two target genes-HvTIR1 and HvAFB-through a modified form of 5'-RACE (rapid amplification of cDNA ends) as well as degradome data analysis. Furthermore, we investigated the biological function of the miR393/target module in root development and its Al stress response. The investigation showed that miR393 affected root growth and adventitious root number by altering auxin sensitivity. Al3+ exposure suppressed miR393 expression in root apex, while overexpression of miR393 counteracted Al-induced inhibition of root elongation and alleviated reactive oxygen species (ROS)-induced cell death. Target mimic (MIM393)-mediated inhibition of miR393's activity enhanced root sensitivity to Al toxicity. We also confirmed that auxin enhanced Al-induced root growth inhibition in barley via application of exogenous 1-naphthaleneacetic acid (NAA), and the expression of auxin-responsive genes in the root apex was induced upon Al treatment. Overexpression of miR393 attenuated the effect of exogenous NAA on Al-induced root growth inhibition, and down-regulated the expression of auxin-responsive genes under Al stress, implying that miR393 regulates root sensitivity to Al through the alteration of auxin signaling output in barley. Therefore, these data indicate that miR393 acts as an integrator of environmental cues in auxin signaling, and suggest a new strategy to improve plant resistance to Al toxicity.

microRNAs participate in gene expression regulation and phytohormone cross-talk in barley embryo during seed development and germination.

BACKGROUND: Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. However, detailed spatial and temporal studies of miRNA-mediated regulation, which can reflect links between seed development and germination are still lacking. RESULTS: In this study, we extended our investigation on miRNAs-involved gene regulation by a combined analysis of seed maturation and germination in barley. Through bioinformatics analysis of small RNA sequencing data, a total of 1324 known miRNA families and 448 novel miRNA candidates were identified. Of those, 16 known miRNAs with 40 target genes, and three novel miRNAs with four target genes were confirmed based on degradome sequencing data. Conserved miRNA families such as miR156, miR168, miR166, miR167, and miR894 were highly expressed in embryos of developing and germinating seeds. A barley-specific miRNA, miR5071, which was predicted to target an OsMLA10-like gene, accumulated at a high level, suggesting its involvement in defence response during these two developmental stages. Based on target prediction and Kyoto Encyclopedia of Genes and Genomes analysis of putative targets, nine highly expressed miRNAs were found to be related to phytohormone signalling and hormone cross-talk. Northern blot and qRT-PCR analysis showed that these miRNAs displayed differential expression patterns during seed development and germination, indicating their different roles in hormone signalling pathways. In addition, we showed that miR393 affected seed development through targeting two genes encoding the auxin receptors TIR1/AFBs in barley, as over-expression of miR393 led to an increased length-width ratio of seeds, whereas target mimic (MIM393)-mediated inhibition of its activity decreased the 1000-grain weight of seeds. Furthermore, the expression of auxin-responsive genes, abscisic acid- and gibberellic acid-related genes was altered in miR393 misexpression lines during germination and early seedling growth. CONCLUSIONS: Our work indicates that miRNA-target pairs participate in gene expression regulation and hormone interaction in barley embryo and provides evidence that miR393-mediated auxin response regulation affects grain development and influences gibberellic acid and abscisic acid homeostasis during germination.

Clathrin regulates blue light-triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis.

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP-binding cassette B19 (ABCB19) and PIN-formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL-triggered, PIN3-mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL-triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N-1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid affect BL-induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL-triggered hypocotyl phototropism in Arabidopsis.

Over-expression of (1,3;1,4)-ß-D-glucanase isoenzyme EII gene results in decreased (1,3;1,4)-ß-D-glucan content and increased starch level in barley grains.

BACKGROUND: High content of (1,3;1,4)-ß-d-glucan in barley grains is regarded as an undesirable factor affecting malting potential, brewing yield and feed utilization. Production of thermostable bacterial (1,3;1,4)-ß-glucanase in transgenic barley grain or supplementation of exogenous bacterial (1,3;1,4)-ß-glucanase has been used to improve malt and feed quality. The aim of the present study was to investigate the effect of over-expression of an endogenous (1,3;1,4)-ß-glucanase on ß-glucan content and grain composition in barley. RESULTS: A construct containing full-length HvGlb2 cDNA encoding barley (1,3;1,4)-ß-glucanase isoenzyme EII under the control of a promoter of barley D-Hordein gene Hor3-1 was introduced into barley cultivar Golden Promise via Agrobacterium-mediated transformation, and transgenic plants were regenerated after hygromycin selection. The T2 generation of proHor3:HvGlb2 transgenic lines showed increased activity of (1,3;1,4)-ß-glucanase in grains. Total ß-glucan content was reduced by more than 95.73% in transgenic grains compared with the wild-type control. Meanwhile, over-expression of (1,3;1,4)-ß-glucanase led to an increase in 1000-grain weight, which might be due to elevated amounts of starch in the grain. CONCLUSION: Manipulating the expression of (1,3;1,4)-ß-glucanase EII can control the ß-glucan content in grain with no apparent harmful effects on grain quality of transgenic plants. © 2016 Society of Chemical Industry.

The miR393a/target module regulates seed germination and seedling establishment under submergence in rice (Oryza sativa L.).

The conserved miRNA393 family is thought to be involved in root elongation, leaf development and stress responses, but its role during seed germination and seedling establishment remains unclear. In this study, expression of the MIR393a/target module and its role in germinating rice (Oryza sativa L.) seeds were investigated. ß-Glucuronidase (GUS) analysis showed that MIR393a and OsTIR1 had spatial-temporal transcriptional activities in radicle roots, coleoptile tips and stomata cells, corresponding to a dynamic auxin response. miR393a promoted primary root elongation when rice seeds were germinated in air and inhibited coleoptile elongation and stomatal development when seeds were submerged. Under submergence, the expression of miR393a was inhibited, and then the auxin response was induced. In the process, OsTIR1 and OsAFB2, auxin receptor genes, were negatively regulated by miR393. We found that miR393a inhibited stomatal development and coleoptile elongation but promoted free indole acetic acid (IAA) accumulation in the rice coleoptile tips. In addition, exogenous abscisic acid (ABA) enhanced the expression of miR393 and inhibited coleoptile growth. Together, miR393a/target plays a role in coleoptile elongation and stomatal development via modulation of auxin signalling during seed germination and seedling establishment under submergence. This study provides new perspectives on the direct sowing of rice seeds in flooded paddy fields.

[The regulatory roles of small RNAs in phytohormone signaling pathways].

Phytohormones are signaling molecules that control plant growth and development. Recent studies revealed that non-coding small RNAs play critical roles in plant development and stress responses via phytohormone signaling pathways. In this review, we summarize the present knowledge on the microRNAs (miRNAs) and secondary short interfering RNAs (siRNAs) involved in phytohormone signaling pathways, which include auxin, gibberellic acid, brassinosteroid and abscisic acid pathways. We also discuss their possible implications in phytohormone crosstalk during specific developmental processes.

Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.

Progress on the autophagic regulators and receptors in plants.

Autophagy is an evolutionarily highly conserved catabolic pathway among eukaryotic cells that protects the organisms against environmental stress. Normally, autophagy is mainly involved with autophagy-related proteins(ATGs) and autophagic regulators including a series of cytoplasmic proteins and small molecules. Besides, the selective autophagy, which targets damaged organalles or protein aggregates, is mediated by the additional receptors to help the ATGs recognize different substrates. In this review, we summarize recent advances in autophagic regulators like ROS(Reactive oxygen species), TOR(Target of rapamycin) and receptors like NBR1(Neighbor of BRCA1 gene protein), RPN10(Regulatory particle non-ATPase 10) as well as their functional mechanisms mainly in Arabidopsis thaliana.

miR396a-Mediated basic helix-loop-helix transcription factor bHLH74 repression acts as a regulator for root growth in Arabidopsis seedlings.

miR396 targets seven GROWTH-REGULATING FACTOR (GRF) genes and the BASIC HELIX-LOOP-HELIX (bHLH) TRANSCRIPTION FACTOR 74 gene (bHLH74) in Arabidopsis. Previous research revealed that the miR396 target module regulates cell proliferation and plays a critical role in leaf development. However, no additional biological functions of miR396 have been investigated in detail. In this study, T-DNA insertion mutants and transgenic plants with altered levels of miR396 or its target genes were used to characterize the regulatory role of miR396 in root development. We found that AtMIR396a was the predominant source for miR396 accumulation in the roots of seedlings, and that the mir396a-1 mutant had longer roots than wild-type seedlings. Overexpression of AtMIR396a decreased the transcript levels of target genes such as GRF genes and bHLH74, and resulted in a shorter root phenotype. Furthermore, the bhlh74-1 mutant had shorter roots, whereas overexpression of an miR396-resistant form of bHLH74 (mbHLH74) had an enhanced root growth phenotype. Moreover, MIR396a regulated root growth by affecting the elongation zone. Taken together, these data indicate that miR396a-mediated bHLH74 repression helps regulate root growth in Arabidopsis seedlings.

Heterologous expression and purification of dermaseptin S4 fusion in Escherichia coli and recovery of biological activity.

Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system.

Functions of miR126 and innate immune response.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level by base pairing with target mRNAs. Many miRNAs have been proven to regulate a broad range of processes, including proliferation, differentiation, development and apoptosis. miR126 is encoded by intron 7 of the EGF- like domain 7 (Egfl7) gene and highly expressed in human endothelial cells (ECs) and plasmacytoid dendritic cells (pDCs). Based on the current knowledge, miR126 is involved in angiogenesis and cancer. Also miR126 is the first miRNA which has been reported to involve in preconditioning the responsiveness of the host to pathogen infection in the steady state. This implicated that miR126 may be the potential therapeutic target for cancer and autoimmune diseases. In this review, we summarize the functions of miR126 in angiogenesis and cancer, and emphasize its relationship with innate immune response.