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.

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.

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.

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.

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.

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.

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.

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.

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.

Callus Initiation from Root Explants Employs Different Strategies in Rice and Arabidopsis.

Callus formation in tissue culture follows the rooting pathway, and newly formed callus seems to be a group of root primordium-like cells. However, it is not clear whether there are multiple mechanisms of callus initiation in different species and in different organs. Here we show that the OsIAA11-mediated pathway is specifically and strictly required for callus initiation in the lateral root (LR) formation region of the primary root (PR) but not for callus initiation at the root tip or the stem base in rice. OsIAA11 and its Arabidopsis homolog AtIAA14 are key players in lateral rooting. However, the AtIAA14-mediated pathway is not strictly required for callus initiation in the LR formation region in Arabidopsis. LRs can be initiated through either the AtIAA14-mediated or AtWOX11-mediated pathway in the Arabidopsis PR, therefore providing optional pathways for callus initiation. In contrast, OsIAA11 is strictly required for lateral rooting in the rice PR, meaning that the OsIAA11 pathway is the only choice for callus initiation. Our study suggests that multiple pathways may converge to WOX5 activation during callus formation in different organs and different species.

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.

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.

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.

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis.

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated expressions of two cysteine desulfhydrases, and exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower endogenous H2S level and decreased stress resistance. Moreover, H2S upregulated the transcripts of multiple abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly, MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines. Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis, and MIR393-targeted auxin receptors.