Switching action modes of miR408-5p mediates auxin signaling in rice.

MicroRNAs (miRNAs) play fundamental roles in many developmental and physiological processes in eukaryotes. MiRNAs in plants generally regulate their targets via either mRNA cleavage or translation repression; however, which approach plays a major role and whether these two function modes can shift remains elusive. Here, we identify a miRNA, miR408-5p that regulates AUXIN/INDOLE ACETIC ACID 30 (IAA30), a critical repressor in the auxin pathway via switching action modes in rice. We find that miR408-5p usually inhibits IAA30 protein translation, but in a high auxin environment, it promotes the decay of IAA30 mRNA when it is overproduced. We further demonstrate that IDEAL PLANT ARCHITECTURE1 (IPA1), an SPL transcription factor regulated by miR156, mediates leaf inclination through association with miR408-5p precursor promoter. We finally show that the miR156-IPA1-miR408-5p-IAA30 module could be controlled by miR393, which silences auxin receptors. Together, our results define an alternative auxin transduction signaling pathway in rice that involves the switching of function modes by miR408-5p, which contributes to a better understanding of the action machinery as well as the cooperative network of miRNAs in plants.

Coordinated Transcriptome and Metabolome Analyses of a Barley hvhggt Mutant Reveal a Critical Role of Tocotrienols in Endosperm Starch Accumulation.

Tocotrienols and tocopherols (vitamin E) are potent antioxidants that are synthesized in green plants. Unlike ubiquitous tocopherols, tocotrienols predominantly accumulate in the endosperm of monocot grains, catalyzed by homogentiate geranylgeranyl transferase (HGGT). Previously, we generated a tocotrienol-deficient hvhggt mutant with shrunken barley grains. However, the relationship between tocotrienols and grain development remains unclear. Here, we found that the hvhggt lines displayed hollow endosperms with defective transfer cells and reduced aleurone layers. The carbohydrate and starch contents of the hvhggt endosperm decreased by approximately 20 and 23%, respectively. Weighted gene coexpression network analyses identified a critical gene module containing HvHGGT, which was strongly associated with the hvhggt mutation and enriched with gene functions in starch and sucrose metabolism. Metabolome measurements revealed an elevated soluble sugar content in the hvhggt endosperm, which was significantly associated with the identified gene modules. The hvhggt endosperm had significantly higher NAD(H) and NADP(H) contents and lower levels of ADPGlc (regulated by redox balance) than the wild-type, consistent with the absence of tocotrienols. Interestingly, exogenous α-tocotrienol spraying on developing hvhggt spikes partially rescued starch accumulation and endosperm defects. Our study supports a potential novel function of tocotrienols in grain starch accumulation and endosperm development in monocot crops.

A unique mutation in PIN-FORMED1 and a genetic pathway for reduced sensitivity of Arabidopsis roots to N-1-naphthylphthalamic acid.

The small chemical N-1-naphthylphthalamic acid (NPA) has long been used as a polar auxin transport inhibitor. Recent biochemical and structural investigations have revealed that this molecule competes with the auxin IAA (indole-3-acetic acid) inside the PIN-FORMED auxin efflux carriers. However, the existence of any mutations in PIN family proteins capable of uncoupling the docking of IAA from NPA remains unclear. We report that Arabidopsis thaliana seedlings overexpressing SMALL AUXIN UP RNA 41 were hypersensitive to NPA-induced root elongation inhibition. We mutagenized this line to improve the genetic screening efficiency for NPA hyposensitivity mutants. Using bulked segregation analysis and mapping-by-sequencing assessment of these mutants, we identified a core genetic pathway for NPA-induced root elongation inhibition, including genes required for auxin biosynthesis, transportation, and signaling. To evaluate specific changes of auxin signaling activity in mutant roots before and after NPA treatment, the DR5::GFP/DR5::YFP markers were introduced and observed. Most importantly, we discovered a unique mutation in the PIN1 protein, substituting a proline residue with leucine at position 584, leading to a loss of NPA sensitivity while keeping the auxin efflux capacity. Transforming the null mutant pin1-201 with the PIN1::PIN1P584L -GFP fusion construct rescued the PIN1 function and provided NPA hyposensitivity. The proline residue is predicted to be adjacent to a hinge in the middle region of the ninth transmembrane helix of PIN1 and is conserved from moss to higher plants. Our work may bring new insights into the engineering of NPA-resistant PINs for auxin biology studies.

Transcriptional and protein structural characterization of homogentisate phytyltransferase genes in barley, wheat, and oat.

BACKGROUND: Homogentisate phytyltransferase (HPT) is the critical enzyme for the biosynthesis of tocopherols (vitamin E), which are the major lipid-soluble antioxidants and help plants adapt to various stress conditions. HPT is generally strictly conserved in various plant genomes; however, a divergent lineage HPT2 was identified recently in some Triticeae species. The molecular function and transcriptional profiles of HPT2 remain to be characterized. RESULTS: In this study, we performed comprehensive transcriptome data mining of HPT1 and HPT2 in different tissues and stages of barley (Hordeum vulgare), wheat (Triticum aestivum), and oat (Avena sativa), followed by qRT-PCR experiments on HPT1 and HPT2 in different tissues of barley and wheat. We found that the common HPT1 genes (HvHPT1, TaHPT1s, and AsHPT1s) displayed a conserved transcriptional pattern in the three target species and were universally transcribed in various tissues, with a notable preference in leaf. In contrast, HPT2 genes (HvHPT2, TaHPT2, and AsHPT2) were specifically transcribed in spike (developmentally up-regulated) and shoot apex tissues, displaying a divergent tissue-specific pattern. Cis-regulatory elements prediction in the promoter region identified common factors related to light-, plant hormone-, low temperature-, drought- and defense- responses in both HPT1s and HPT2s. We observed the transcriptional up-regulation of HvHPT1 and HvHPT2 under various stress conditions, supporting their conserved function in environmental adaption. We detected a clear, relaxed selection pressure in the HPT2 lineage, consistent with the predicted evolution pattern following gene duplication. Protein structural modelling and substrate docking analyses identified putative catalytic amino acid residues for HvHPT1 and HvHPT2, which are strictly conserved and consistent with their function in vitamin E biosynthesis. CONCLUSIONS: We confirmed the presence of two lineages of HPT in Triticeae and Aveninae, including hexaploid oat, and characterized their transcriptional profiles based on transcriptome and qRT-PCR data. HPT1s were ubiquitously transcribed in various tissues, whilst HPT2s were highly expressed in specific stages and tissue. The active transcription of HPT2s, together with its conserved cis-elements and protein structural features, support HPT2s' role in tocopherol production in Triticeae. This study is the first protein structural analysis on the membrane-bound plant HPTs and provides valuable insights into its catalytic mechanism.

Initiation of scutellum-derived callus is regulated by an embryo-like developmental pathway in rice.

In rice (Oryza sativa) tissue culture, callus can be induced from the scutellum in embryo or from the vasculature of non-embryonic organs such as leaves, nodes, or roots. Here we show that the auxin signaling pathway triggers cell division in the epidermis of the scutellum to form an embryo-like structure, which leads to callus formation. Our transcriptome data show that embryo-, stem cell-, and auxin-related genes are upregulated during scutellum-derived callus initiation. Among those genes, the embryo-specific gene OsLEC1 is activated by auxin and involved in scutellum-derived callus initiation. However, OsLEC1 is not required for vasculature-derived callus initiation from roots. In addition, OsIAA11 and OsCRL1, which are involved in root development, are required for vasculature-derived callus formation but not for scutellum-derived callus formation. Overall, our data indicate that scutellum-derived callus initiation is regulated by an embryo-like development program, and this is different from vasculature-derived callus initiation which borrows a root development program.

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice.

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin (OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines. Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways. We also suggested rice auxin response factor (OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the 'best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

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.

Rice LEAFY COTYLEDON1 Hinders Embryo Greening During the Seed Development.

LEAFY COTYLEDON1 (LEC1) is the central regulator of seed development in Arabidopsis, while its function in monocots is largely elusive. We generated Oslec1 mutants using CRISPR/Cas9 technology. Oslec1 mutant seeds lost desiccation tolerance and triggered embryo greening at the early development stage. Transcriptome analysis demonstrated that Oslec1 mutation altered diverse hormonal pathways and stress response in seed maturation, and promoted a series of photosynthesis-related genes. Further, genome-wide identification of OsLEC1-binding sites demonstrated that OsLEC1 bound to genes involved in photosynthesis, photomorphogenesis, as well as abscisic acid (ABA) and gibberellin (GA) pathways, involved in seed maturation. We illustrated an OsLEC1-regulating gene network during seed development, including the interconnection between photosynthesis and ABA/GA biosynthesis/signaling. Our findings suggested that OsLEC1 acts as not only a central regulator of seed maturation but also an inhibitor of embryo greening during rice seed development. This study would provide new understanding for the OsLEC1 regulatory mechanisms on photosynthesis in the monocot seed development.

Transcriptional landscapes of de novo root regeneration from detached Arabidopsis leaves revealed by time-lapse and single-cell RNA sequencing analyses.

Detached Arabidopsis thaliana leaves can regenerate adventitious roots, providing a platform for studying de novo root regeneration (DNRR). However, the comprehensive transcriptional framework of DNRR remains elusive. Here, we provide a high-resolution landscape of transcriptome reprogramming from wound response to root organogenesis in DNRR and show key factors involved in DNRR. Time-lapse RNA sequencing (RNA-seq) of the entire leaf within 12 h of leaf detachment revealed rapid activation of jasmonate, ethylene, and reactive oxygen species (ROS) pathways in response to wounding. Genetic analyses confirmed that ethylene and ROS may serve as wound signals to promote DNRR. Next, time-lapse RNA-seq within 5 d of leaf detachment revealed the activation of genes involved in organogenesis, wound-induced regeneration, and resource allocation in the wounded region of detached leaves during adventitious rooting. Genetic studies showed that BLADE-ON-PETIOLE1/2, which control aboveground organs, PLETHORA3/5/7, which control root organogenesis, and ETHYLENE RESPONSE FACTOR115, which controls wound-induced regeneration, are involved in DNRR. Furthermore, single-cell RNA-seq data revealed gene expression patterns in the wounded region of detached leaves during adventitious rooting. Overall, our study not only provides transcriptome tools but also reveals key factors involved in DNRR from detached Arabidopsis leaves.

Function, Mechanism, and Application of Plant Melatonin: An Update with a Focus on the Cereal Crop, Barley (Hordeum vulgare L.).

Melatonin is a multiple-function molecule that was first identified in animals and later in plants. Plant melatonin regulates versatile processes involved in plant growth and development, including seed germination, root architecture, flowering time, leaf senescence, fruit ripening, and biomass production. Published reviews on plant melatonin have been focused on two model plants: (1) Arabidopsis and (2) rice, in which the natural melatonin contents are quite low. Efforts to integrate the function and the mechanism of plant melatonin and to determine how plant melatonin benefits human health are also lacking. Barley is a unique cereal crop used for food, feed, and malt. In this study, a bioinformatics analysis to identify the genes required for barley melatonin biosynthesis was first performed, after which the effects of exogenous melatonin on barley growth and development were reviewed. Three integrated mechanisms of melatonin on plant cells were found: (1) serving as an antioxidant, (2) modulating plant hormone crosstalk, and (3) signaling through a putative plant melatonin receptor. Reliable approaches for characterizing the function of barley melatonin biosynthetic genes and to modulate the melatonin contents in barley grains are discussed. The present paper should be helpful for the improvement of barley production under hostile environments and for the reduction of pesticide and fungicide usage in barley cultivation. This study is also beneficial for the enhancement of the nutritional values and healthcare functions of barley in the food industry.

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.

Whole Grain Qingke Attenuates High-Fat Diet-Induced Obesity in Mice With Alterations in Gut Microbiota and Metabolite Profile.

Whole grain Qingke (WGQK) displays anti-obesity and lipid-lowering properties; however, the underlying mechanism remains elusive. This study investigated the alteration of gut microbiota composition and metabolite profile induced by WGQK intervention in mice through the integration of 16S ribosomal RNA (rRNA) sequencing and an untargeted metabolomics study. C57BL/6J male mice were fed a normal control diet (NC), high-fat diet (HFD), and HFD plus 30% WGQK (HFD+QK) for 16 weeks. The WGQK intervention decreased body weight gain, glucose tolerance, and serum lipid levels, and alleviated liver function damage induced by HFD. Moreover, WGQK changed gut microbiota composition and enriched specific genera such as Akkermansia, Bifidobacterium, and Lactobacillus. Fecal metabolomics analysis indicated that WGQK enhanced the abundance of tryptophan metabolism-related metabolites (indole, 3-indoleacetic acid, indole acetic acid (IAA), 5-hydroxyindole-3-acetic acid), histidine metabolism-related metabolites (histamine), and some unsaturated fatty acids (oleic acid, 9,10-dihydroxy-12Z-octadecenoic acid, and alpha-linolenic acid). Spearman correlation analysis revealed that these metabolites were negatively correlated with obesity-related parameters and positively correlated with the gut genera enriched by WGQK. Moreover, WGQK promoted the expression of Cholesterol 7α-hydroxylase (CYP7A1) responsible for primary bile acids production, accompanied by a decline in intestinal FXR-FGF15 expression levels. The transcript levels of two genes associated with lipogenesis, such as lipid fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were also decreased in the HFD+QK group. Overall, our results suggest interactions between gut microbial shifts and host amino acid/lipid metabolism, and shed light on the mechanisms underlying the anti-obesity effect of WGQK.

Manipulating a Single Transcription Factor, Ant1, Promotes Anthocyanin Accumulation in Barley Grains.

Barley has abundant anthocyanin-rich accessions, which renders it an ideal model to investigate the regulatory mechanism of anthocyanin biosynthesis. This study functionally characterized two transcription factors: Ant1 and Ant2. Sequence alignment showed that the coding sequences of Ant1 and Ant2 are conserved among 11 colored hulless barley and noncolored barley varieties. The expression profiles of Ant1 and Ant2 were divergent between species, and significantly higher expression was found in two colored Qingke accessions. The co-expression of Ant1 and Ant2 resulted in purple pigmentation in transient transformation systems via the promotion of the transcription of four structural genes. Ant1 interacted with Ant2, and overexpression of Ant1 activated the transcription of Ant2. Moreover, overexpression of Ant1 led to anthocyanin accumulation in the pericarp and aleurone layer of transgenic barley grains. Overall, our results suggest that anthocyanin-enriched barley grains can be produced by manipulating Ant1 expression.

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 heterochronic gene Oryza sativa LIKE HETEROCHROMATIN PROTEIN 1 modulates miR156b/c/i/e levels.

The juvenile-to-adult transition in plants involves changes in vegetative growth and plant architecture; the timing of this transition has important implications for agriculture. The microRNA miR156 regulates this transition and shoot maturation in plants. In Arabidopsis thaliana, deposition of histone H3 trimethylation on lysine 27 (H3K27me3, a repressive mark) at the MIR156A/C loci is regulated by Polycomb Repressive Complex 1 (PRC1) or PRC2, depending on the developmental stage. The levels of miR156 progressively decline during shoot maturation. The amount of H3K27me3 at MIR156A/C loci affects miR156 levels; however, whether this epigenetic regulation is conserved remains unclear. Here, we found that in rice (Oryza sativa), the putative PRC1 subunit LIKE HETEROCHROMATIN PROTEIN 1 (OsLHP1), with the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module, affects developmental phase transitions. Loss of OsLHP1 function results in ectopic expression of MIR156B/C/I/E, phenocopy of miR156 overexpression, and reduced H3k27me3 levels at MIR156B/C/I/E. This indicates that OsLHP1 has functionally diverged from Arabidopsis LHP1. Genetic and transcriptome analyses of wild-type, miR156b/c-overexpression, and Oslhp1-2 mutant plants suggest that OsLHP1 acts upstream of miR156 and SPL during the juvenile-to-adult transition. Therefore, modifying the OsLHP1-miR156-SPL pathway may enable alteration of the vegetative period and plant architecture.

OsHDA710-Mediated Histone Deacetylation Regulates Callus Formation of Rice Mature Embryo.

Histone deacetylases (HDACs) play important roles in the regulation of eukaryotic gene expression. The role of HDACs in specialized transcriptional regulation and biological processes is poorly understood. In this study, we evaluated the global expression patterns of genes related to epigenetic modifications during callus initiation in rice. We found that the repression of HDAC activity by trichostatin A (TSA) or by OsHDA710 mutation (hda710) results in impaired callus formation of rice mature embryo and increased global histone H3 acetylation levels. The HDAC inhibition decreased auxin response and cell proliferation in callus formation. Meanwhile, the transcriptional repressors OsARF18 and OsARF22 were upregulated in the callus of hda710. The chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis demonstrated that the callus of hda710 exhibited enhanced histone H3 acetylation levels at the chromatin regions of OsARF18 and OsARF22. Furthermore, we found that OsARF18 and OsARF22 were regulated through OsHDA710 recruitment to their target loci. In addition, overexpression of OsARF18 decreased the transcription of downstream genes PLT1 and PLT2 and inhibited callus formation of the mature embryo. These results demonstrate that OsHDA710 regulates callus formation by suppressing repressive OsARFs via histone deacetylation during callus formation of rice mature embryo. This indicates that OsHDA710-mediated histone deacetylation is an epigenetic regulation pathway for maintaining auxin response during cell dedifferentiation.

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.

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.

The barley miR393 has multiple roles in regulation of seedling growth, stomatal density, and drought stress tolerance.

microRNA393 (miR393) and its target module have been implicated as comprising a conserved mechanism to regulate developmental processes and plant growth in response to environmental signals through the auxin signaling pathway. Our previous work identified miR393 and its two targets in barley. In this study, we further investigated the expression pattern of miR393 and its biological functions in seedling growth and drought tolerance. We showed that the miR393 overexpressing line (OE) exhibited increased stomatal density with decreased guard cell length, while the miR393 knockdown line (MIM) displayed the opposite phenotype, which might be due to the effects of miR393 on AUXIN RESPONSE FACTOR5 (ARF5) and three stomatal development-related genes, such as EPIDERMAL PATTERNING FACTOR1 (EPF1), SPEECHLESS (SPCH), and MUTE. In addition, the MIM line conferred enhanced drought tolerance, with alleviated leaf chlorosis and lipid peroxidation after 22 days drought treatment. In contrast, the OE line was more sensitive to drought stress and accumulated more malondialdehyde and hydrogen peroxide than the wild type. Furthermore, polyethylene glycol (PEG) treatment-induced abscisic acid (ABA) accumulation in leaves was suppressed in the OE line, indicating that miR393 might regulate drought stress response and tolerance through its interaction with ABA biosynthesis. Overall, these data suggest that miR393 might be a potential target for manipulation of stomatal density and improvement of drought tolerance in barley.