SQUAMOSA PROMOTER BINDING-LIKE Research Articles

Natural variation in CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 reveals a novel role of calcium signaling in vegetative phase change in Arabidopsis.

The timing of vegetative phase change (VPC) in plants is regulated by a temporal decline in the expression of miR156. Both exogenous cues and endogenous factors, such as temperature, light, sugar, nutrients, and epigenetic regulators, have been shown to affect VPC by altering miR156 expression. However, the genetic basis of natural variation in VPC remains largely unexplored. Here, we conducted a genome-wide association study on the variation of the timing of VPC in Arabidopsis. We identified CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 (CNGC4) as a significant locus associated with the diversity of VPC. Mutations in CNGC4 delayed VPC, accompanied by an increased expression level of miR156 and a corresponding decrease in SQUAMOSA PROMOTER BINDING-LIKE (SPL) gene expression. Furthermore, mutations in CNGC2 and CATION EXCHANGER 1/3 (CAX1/3) also led to a delay in VPC. Polymorphisms in the CNGC4 promoter contribute to the natural variation in CNGC4 expression and the diversity of VPC. Specifically, the early CNGC4 variant promotes VPC and enhances plant adaptation to local environments. In summary, our findings offer genetic insights into the natural variation in VPC in Arabidopsis, and reveal a previously unidentified role of calcium signaling in the regulation of VPC.

How to awaken a sleeping giant: antagonistic expression of Flowering locus T homologs and elements of the age-related pathway are associated with the flowering transition in Agave tequilana

Key messageAntagonistic expression of Flowering locus T proteins and the ageing pathway via miRNAs and sugar metabolism regulate the initiation of flowering in A. tequilana.Flowering in commercial plantations of Agave tequilana signals that plants are ready to harvest for tequila production. However, time of flowering is often unpredictable and a detailed understanding of the process would be beneficial in the field, for breeding and for the development of future research. This report describes the functional analysis of A. tequilana FLOWERING LOCUS T (FT) genes by heterologous expression in A. thaliana and in situ hybridization in agave plants. The gene structures of the Agave tequilana FT family are also described and putative regulatory promoter elements were identified. Most Agave species have monocarpic, perennial life cycles that can last over 25 years during which plants do not respond to the normal environmental signals which induce flowering, suggesting that the ageing pathway as described in Arabidopsis may play an important role in determining flowering time in these species. Elements of this pathway were analyzed and in silico data is presented that supports the regulation of SQUAMOSA PROMOTER BINDING LIKE proteins (SPL), APETALA2 (AP2) proteins and members of Plant Glycoside Hydrolase Family 32 (PGHF32) by interactions with miRNAs 156, 172 and 164 during the initiation of flowering in A. tequilana.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

Functional conservation and divergence of miR156 and miR529 during rice development

MicroRNAs (miRNAs) are important regulatory elements involved in the regulation of various plant developmental and physiological processes by blocking the expression of target genes. MiR156 and miR529 are two combinatorial regulators, which cooperatively target the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family genes. However, there has been no report about the functional conservation and divergence of miR156 and miR529 during plant development to date. In this study, the biological function and relationship of miR156, miR529 and their target OsSPL14 in rice were explored. Overexpression of miR156e or miR529a (miR156e-OE and miR529a-OE) increased the grain size and tiller number but decreased the plant height and panicle length, while an opposite phenotype was observed for their target mimicry (miR156-MIMIC and miR529a-MIMIC) transgenic plants. Stem-loop RT-PCR results revealed ubiquitous expression of miR156 in roots, axillary buds and leaves, while miR529 was preferentially expressed in the panicle. Accordingly, OsSPL14 could be preferentially and precisely cleaved by miR529a in young panicle but by miR156 in vegetative tissues. Transgenic plants generated by the target immune strategy exhibited obvious growth defects upon the blocking of miR156 and/or miR529 function in rice, confirming that both miR156 and miR529 play important roles in controlling rice growth and development. Moreover, the miR156/miR529-OsSPL14 module negatively controlled grain size by regulating the genes associated with grain size and cell cycling, and controlled plant height through a more complicated mechanism. Taken together, our results demonstrate that miR156 and miR529 respectively function dominantly in the vegetative stage and reproductive stage to control rice growth and development by regulating the accumulation of OsSPL14. These findings facilitate a better understanding of the functional conservation and divergence of miR156 and miR529 family in the miRNA combinatorial regulatory network of plants.

Role of a ZF-HD Transcription Factor in miR157-Mediated Feed-Forward Regulatory Module That Determines Plant Architecture in Arabidopsis.

In plants, vegetative and reproductive development are associated with agronomically important traits that contribute to grain yield and biomass. Zinc finger homeodomain (ZF-HD) transcription factors (TFs) constitute a relatively small gene family that has been studied in several model plants, including Arabidopsis thaliana L. and Oryza sativa L. The ZF-HD family members play important roles in plant growth and development, but their contribution to the regulation of plant architecture remains largely unknown due to their functional redundancy. To understand the gene regulatory network controlled by ZF-HD TFs, we analyzed multiple loss-of-function mutants of ZF-HD TFs in Arabidopsis that exhibited morphological abnormalities in branching and flowering architecture. We found that ZF-HD TFs, especially HB34, negatively regulate the expression of miR157 and positively regulate SQUAMOSA PROMOTER BINDING–LIKE 10 (SPL10), a target of miR157. Genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis revealed that miR157D and SPL10 are direct targets of HB34, creating a feed-forward loop that constitutes a robust miRNA regulatory module. Network motif analysis contains overrepresented coherent type IV feedforward motifs in the amiR zf-HD and hbq mutant background. This finding indicates that miRNA-mediated ZF-HD feedforward modules modify branching and inflorescence architecture in Arabidopsis. Taken together, these findings reveal a guiding role of ZF-HD TFs in the regulatory network module and demonstrate its role in plant architecture in Arabidopsis.

Genome-wide identification and characterization of the SPL gene family and its expression in the various developmental stages and stress conditions in foxtail millet (Setaria italica)

BackgroundAmong the major transcription factors, SPL plays a crucial role in plant growth, development, and stress response. Foxtail millet (Setaria italica), as a C4 crop, is rich in nutrients and is beneficial to human health. However, research on the foxtail millet SPL (SQUAMOSA PROMOTER BINDING-LIKE) gene family is limited. ResultsIn this study, a total of 18 SPL genes were identified for the comprehensive analysis of the whole genome of foxtail millet. These SiSPL genes were divided into seven subfamilies (I, II, III, V, VI, VII, and VIII) according to the classification of the Arabidopsis thaliana SPL gene family. Structural analysis of the SiSPL genes showed that the number of introns in subfamilies I and II were much larger than others, and the promoter regions of SiSPL genes were rich in different cis-acting elements. Among the 18 SiSPL genes, nine genes had putative binding sites with foxtail millet miR156. No tandem duplication events were found between the SiSPL genes, but four pairs of segmental duplications were detected. The SiSPL genes expression were detected in different tissues, which was generally highly expressed in seeds development process, especially SiSPL6 and SiSPL16, which deserve further study. The results of the expression levels of SiSPL genes under eight types of abiotic stresses showed that many stress responsive genes, especially SiSPL9, SiSPL10, and SiSPL16, were highly expressed under multiple stresses, which deserves further attention.ConclusionsIn this research, 18 SPL genes were identified in foxtail millet, and their phylogenetic relationships, gene structural features, duplication events, gene expression and potential roles in foxtail millet development were studied. The findings provide a new perspective for the mining of the excellent SiSPL gene and the molecular breeding of foxtail millet.

Defining the components of the miRNA156-SPL-miR172 aging pathway in pea and their expression relative to changes in leaf morphology

The timing of developmental phase transitions is crucial for plant reproductive success, and two microRNAs (miRNA), miR156 and miR172, are implicated in the control of these changes, together with their respective SQUAMOSA promoter binding–like (SPL) and APETALA2-like (AP2) targets. While their patterns of regulation have been studied in a growing range of species, to date they have not been examined in pea (Pisum sativum), an important legume crop and model species. We analysed the recently released pea genome and defined nine miR156, 21 SPL, four miR172, and five AP2-like genes. Phylogenetic analysis of the SPL genes in pea, Medicago and Arabidopsis confirmed the eight previously defined clades, and identified a ninth potentially legume-specific SPL clade in pea and Medicago. Among the PsSPL, 14 contain a miR156 binding site and all five AP2-like transcription factors in pea include a miR172 binding site. Phylogenetic relationships, expression levels and temporal expression changes identified PsSPL2a/3a/3c/6b/9a/9b/13b/21, PsmiR156d/j and PsmiR172a/d as the most likely of these genes to participate in phase change in pea. Comparisons with leaf morphology suggests that vegetative phase change is unlikely to be definitively marked by a change in leaflet number. In addition, the timing of FT gene induction suggests that the shift from the juvenile to the adult vegetative phase may occur within fourteen days in plants grown under inductive conditions, and calls into question the contribution of miR172/AP2 to the floral transition. This work provides the first insight into the nature of vegetative phase change in pea, and an important foundation for future functional studies.

Transcriptional profile of AvrRpt2EA-mediated resistance and susceptibility response to Erwinia amylovora in apple

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor.

CRISPR-based assessment of genomic structure in the conserved SQUAMOSA promoter-binding-like gene clusters in rice.

Although SQUAMOSA promoter-binding-like (SPL) transcription factors are important regulators of development in rice (Oryza sativa), prior assessments of the SPL family have been limited to single genes. A functional comparison across the full gene family in standardized genetic backgrounds has not been reported previously. Here, we demonstrate that the SPL gene family in rice is enriched due to the most recent whole genome duplication (WGD). Notably, 10 of 19 rice SPL genes (52%) cluster in four units that have persisted for at least 50 million years. We show that SPL gene grouping and retention following WGD is widespread in angiosperms, suggesting the conservatism and importance of this gene arrangement. We used Cas9 editing to generate transformation lines for all 19 SPL genes in a common set of backgrounds, and found that knockouts of 14 SPL genes exhibited defects in plant height, 10 exhibited defects in panicle size, and nine had altered grain lengths. We observed subfunctionalization of genes in the paleoduplicated pairs, but little evidence of neofunctionalization. Expression of OsSPL3 was negatively correlated with that of its closest neighbor in its synteny group, OsSPL4, and its sister paired gene, OsSPL12, in the opposing group. Nucleotide diversity was lower in eight of the nine singleton genes in domesticated rice, relative to wild rice, whereas the reverse was true for the paired genes. Together, these results provide functional information on eight previously unexamined OsSPL family members and suggest that paleoduplicate pair redundancy benefits plant survival and innovation.

MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.)

Rice is one of the most important food crops in the world. Breeding high-yield, multi-resistant and high-quality varieties has always been the goal of rice breeding. Rice tiller, panicle architecture and grain size are the constituent factors of yield, which are regulated by both genetic and environmental factors, including miRNAs, transcription factors, and downstream target genes. Previous studies have shown that SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factors can control rice tiller, panicle architecture and grain size, which were regulated by miR156, miR529 and miR535. In this study, we obtained miR529a target mimicry (miR529a-MIMIC) transgenic plants to investigate plant phenotypes, physiological and molecular characteristics together with miR529a overexpression (miR529a-OE) and wild type (WT) to explore the function of miR529a and its SPL target genes in rice. We found that OsSPL2, OsSPL17 and OsSPL18 at seedling stage were regulated by miR529a, but there had complicated mechanism to control plant height. OsSPL2, OsSPL16, OsSPL17 and SPL18 at tillering stage were regulated by miR529a to control plant height and tiller number. And panicle architecture and grain size were controlled by miR529a through altering the expression of all five target genes OsSPL2, OsSPL7, OsSPL14, OsSPL16, OsSPL17 and OsSPL18. Our study suggested that miR529a might control rice growth and development by regulating different SPL target genes at different stages, which could provide a new method to improve rice yield by regulating miR529a and its SPL target genes.

An Integrative Analysis of Transcriptome, Proteome and Hormones Reveals Key Differentially Expressed Genes and Metabolic Pathways Involved in Flower Development in Loquat.

Flower development is a vital developmental process in the life cycle of woody perennials, especially fruit trees. Herein, we used transcriptomic, proteomic, and hormone analyses to investigate the key candidate genes/proteins in loquat (Eriobotrya japonica) at the stages of flower bud differentiation (FBD), floral bud elongation (FBE), and floral anthesis (FA). Comparative transcriptome analysis showed that differentially expressed genes (DEGs) were mainly enriched in metabolic pathways of hormone signal transduction and starch and sucrose metabolism. Importantly, the DEGs of hormone signal transduction were significantly involved in the signaling pathways of auxin, gibberellins (GAs), cytokinin, ethylene, abscisic acid (ABA), jasmonic acid, and salicylic acid. Meanwhile, key floral integrator genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and floral meristem identity genes SQUAMOSA PROMOTER BINDING LIKE (SPL), LEAFY (LFY), APETALA1 (AP1), and AP2 were significantly upregulated at the FBD stage. However, key floral organ identity genes AGAMOUS (AG), AP3, and PISTILLATA (PI) were significantly upregulated at the stages of FBE and FA. Furthermore, transcription factors (TFs) such as bHLH (basic helix-loop-helix), NAC (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2) and cup-shaped cotyledon (CUC2)), MYB_related (myeloblastosis_related), ERF (ethylene response factor), and C2H2 (cysteine-2/histidine-2) were also significantly differentially expressed. Accordingly, comparative proteomic analysis of differentially accumulated proteins (DAPs) and combined enrichment of DEGs and DAPs showed that starch and sucrose metabolism was also significantly enriched. Concentrations of GA3 and zeatin were high before the FA stage, but ABA concentration remained high at the FA stage. Our results provide abundant sequence resources for clarifying the underlying mechanisms of the flower development in loquat.

ARGONAUTE5 Represses Age-Dependent Induction of Flowering through Physical and Functional Interaction with miR156 in Arabidopsis

Flowering time is a finely tuned process in plants, in part controlled by the age-regulated microRNA156 (miR156), which functions by suppressing the transcripts of SQUAMOSA-PROMOTER BINDING LIKE (SPL) transcription factors. ARGONAUTE (AGO) proteins are essential effectors of miRNA-mediated gene regulation. However, which AGO(s) mediate(s) the control of flowering time remains unclear. Here, we demonstrate a role of AGO5 in controlling flowering time by modulating the expression of SPL transcription factors. We show that AGO5 interacts physically and functionally with miR156 and that ago5 mutants present an early flowering phenotype in Arabidopsis. Furthermore, in ago5 mutants, the repression of flowering caused by miR156 overexpression is largely reversed, whereas leaf morphology remains unaffected. Our results thus indicate a specific role for AGO5 in mediating miR156 activity in meristematic, but not vegetative, tissue. As such, our data suggest a spatiotemporal regulation of the miR156 aging pathway mediated through different AGO proteins in different tissues.

Wheat TaSPL8 Modulates Leaf Angle Through Auxin and Brassinosteroid Signaling.

In grass crops, leaf angle is determined by development of the lamina joint, the tissue connecting the leaf blade and sheath, and is closely related to crop architecture and yield. In this study, we identified a mutant generated by fast neutron radiation that exhibited an erect leaf phenotype caused by defects in lamina joint development. Map-based cloning revealed that the gene TaSPL8, encoding a SQUAMOSA PROMOTER BINDING-LIKE (SPL) protein, is deleted in this mutant. TaSPL8 knock-out mutants exhibit erect leaves due to loss of the lamina joint, compact architecture, and increased spike number especially in high planting density, suggesting similarity with its LIGULESS1 homologs in maize (Zea mays) and rice (Oryza sativa). Hence, LG1 could be a robust target for plant architecture improvement in grass species. Common wheat (Triticum aestivum, 2n = 6× = 42; BBAADD) is an allohexaploid containing A, B, and D subgenomes and the homeologous gene of TaSPL8 from the D subgenome contributes to the length of the lamina joint to a greater extent than that from the A and B subgenomes. Comparison of the transcriptome between the Taspl8 mutant and the wild type revealed that TaSPL8 is involved in the activation of genes related to auxin and brassinosteroid pathways and cell elongation. TaSPL8 binds to the promoters of the AUXIN RESPONSE FACTOR gene and of the brassinosteroid biogenesis gene CYP90D2 and activates their expression. These results indicate that TaSPL8 might regulate lamina joint development through auxin signaling and the brassinosteroid biosynthesis pathway.

SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass.

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses. Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes. Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses. Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.

The Maize Corngrass1 miRNA-Regulated Developmental Alterations Are Restored by a Bacterial ADP-Glucose Pyrophosphorylase in Transgenic Tobacco.

Crop-based bioethanol has raised concerns about competition with food and feed supplies, and technologies for second- and third-generation biofuels are still under development. Alternative feedstocks could fill this gap if they can be converted to biofuels using current sugar- or starch-to-ethanol technologies. The aim of this study was to enhance carbohydrate accumulation in transgenic Nicotiana benthamiana by simultaneously expressing the maize Corngrass1 miRNA (Cg1) and E. coli ADP-glucose pyrophosphorylase (glgC), both of which have been reported to enhance carbohydrate accumulation in planta. Our findings revealed that expression of Cg1 alone increased shoot branching, delayed flowering, reduced flower organ size, and induced loss of fertility. These changes were fully restored by coexpressing Escherichia coli glgC. The transcript level of miRNA156 target SQUAMOSA promoter binding-like (SPL) transcription factors was suppressed severely in Cg1-expressing lines as compared to the wild type. Expression of glgC alone or in combination with Cg1 enhanced biomass yield and total sugar content per plant, suggesting the potential of these genes in improving economically important biofuel feedstocks. A possible mechanism of the Cg1 phenotype is discussed. However, a more detailed study including genome-wide transcriptome and metabolic analysis is needed to determine the underlying genetic elements and pathways regulating the observed developmental and metabolic changes.

Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley

The SQUAMOSA-promoter binding like (SPL) gene family encodes transcription factors that have been shown in many species to influence plant growth and development, but information about these genes in barley (Hordeum vulgare L.) is limited. This study identified 17 barley SPL genes, within eight distinct groups, that are orthologs of SPL genes described in Arabidopsis, wheat, and rice. Sixteen barley SPLs undergo alternative splicing. Seven SPLs contain a putative miR156 target site and the transcript levels of the miR156-targeted HvSPLs (HvSPL3, 13 and 23) were lower in vegetative than in reproductive phase but this was true also for some SPL genes such as HvSPL6 that were not regulated by miR156. Because SPL gene products regulate miR172, which is also involved in floral development, the expression of miR172 was studied. An antagonistic expression pattern of miR156 and miR172b during the vegetative and the reproductive phases signifies their apparent function in barley growth phase transition. Characterization of a barley mir172 mutant having an abnormal, indeterminate spikelet phenotype suggests the possible feedback role of AP2/miR172 module on HvSPL genes. This is the first comprehensive analysis of the miR156/SPL/miR172 axis in barley that provides a basis to elucidate their roles in various biological processes.

Control of chrysanthemum flowering through integration with an aging pathway

Age, as a threshold of floral competence acquisition, prevents precocious flowering when there is insufficient biomass, and ensures flowering independent of environmental conditions; however, the underlying regulatory mechanisms are largely unknown. In this study, silencing the expression of a nuclear factor gene, CmNF-YB8, from the short day plant chrysanthemum (Chrysanthemum morifolium), results in precocious transition from juvenile to adult, as well as early flowering, regardless of day length conditions. The expression of SQUAMOSA PROMOTER BINDING-LIKE (SPL) family members, SPL3, SPL5, and SPL9, is upregulated in CmNF-YB8-RNAi plants, while expression of the microRNA, cmo-MIR156, is downregulated. In addition, CmNF-YB8 is shown to bind to the promoter of the cmo-MIR156 gene. Ectopic expression of cmo-miR156, using a virus-based microRNA expression system, restores the early flowering phenotype caused by CmNF-YB8 silencing. These results show that CmNF-YB8 influences flowering time through directly regulating the expression of cmo-MIR156 in the aging pathway.

MiR529a modulates panicle architecture through regulating SQUAMOSA PROMOTER BINDING-LIKE genes in rice (Oryza sativa).

MiR529a affects rice panicle architecture by targeting OsSPL2,OsSPL14 and OsSPL17 genes that could regulate their downstream panicle related genes. The panicle architecture determines the grain yield and quality of rice, which could be regulated by many transcriptional factors. The SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors are involved in the regulation of panicle development, which are targeted by miR156 and miR529. The expression profile demonstrated that miR529a is preferentially expressed in the early panicle of rice and it might regulate panicle development in rice. However, the regulation mechanism of miR529-SPL is still not clear. In this study, we predicted five miR529a putative target genes, OsSPL2, OsSPL14, OsSPL16, OsSPL17 and OsSPL18, while only the expression of OsSPL2, OsSPL14, and OsSPL17 was regulated by miR529a in the rice panicle. Overexpression of miR529a dramatically affected panicle architecture, which was regulated by OsSPL2, OsSPL14, and OsSPL17. Furthermore, the 117, 35, and 25 pathway genes associated with OsSPL2, OsSPL14 and OsSPL17, respectively, were predicted, and they shared 20 putative pathway genes. Our results revealed that miR529a could play a vital role in the regulation of panicle architecture through regulating OsSPL2, OsSPL14, OsSPL17 and the complex networks formed by their pathway and downstream genes. These findings will provide new genetic resources for reshaping ideal plant architecture and breeding high yield rice varieties.

Identification and differential regulation of microRNAs in response to methyl jasmonate treatment in Lycoris aurea by deep sequencing

Background Lycoris aurea is a medicine-valuable and ornamental herb widely distributed in China. Former studied have showed that methyl jasmonate (MJ) treatment could increase the content of glanthamine-a worldwide medicine for symptomatic treatment of Alzheimer’s disease in genus Lycoris plants. To explore the possible role of miRNAs in the regulation of jasmonic acid signaling pathway and uncover their potential correlations, we investigated the expression profiles of small RNAs (sRNAs) and their targets in Lycoris aurea, with MJ treatment by using next-generation deep sequencing.ResultsA total of 365 miRNAs were identified, comprising 342 known miRNAs (representing 60 miRNA families) and 23 novel miRNAs. Among them, 143 known and 11 novel miRNAs were expressed differently under MJ treatment. Quantitative real-time PCR of eight selected miRNAs validated the expression pattern of these loci in response to MJ treatment. In addition, degradome sequencing analysis showed that 32 target genes were validated to be targeted by the 49 miRNAs, respectively. Gene function and pathway analyses showed that these targets such as auxin response factors (ARFs), squamosa promoter-binding like (SPL) proteins, basic helix-loop-helix (bHLH) proteins, and ubiquitin-conjugating enzyme E2 are involved in different plant processes, indicating miRNAs mediated regulation might play important roles in L. aurea response to MJ treatment. Furthermore, several L. aurea miRNAs associated with their target genes that might be involved in Amaryllidaceae alkloids biosynthehsis were also analyzed.ConclusionsA number of miRNAs with diverse expression patterns, and complex relationships between expression of miRNAs and targets were identified. This study represents the first transcriptome-based analysis of miRNAs in Lycoris and will contribute to understanding the potential roles of miRNAs involved in regulation of MJ response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2645-y) contains supplementary material, which is available to authorized users.

Profiling and Characterization of Small RNAs in the Liverwort, Marchantia polymorpha, Belonging to the First Diverged Land Plants.

MicroRNAs (miRNAs) have important roles in gene regulation during plant development. Previous studies revealed that some miRNAs are highly shared by most land plants. Recently, the liverwort, Marchantia polymorpha, has been studied by molecular genetic approaches, and sequencing of its genome is currently underway. The expression pattern and the detailed functions of miRNAs during Marchantia development are unknown. Here, we profiled the small RNAs expressed in thalli, antheridiophores and archegoniophores of M. polymorpha using high-throughput RNA sequencing. We revealed that a limited number of miRNAs are shared between M. polymorpha and the moss, Physcomitrella patens, and that a number of miRNAs are M. polymorpha specific. Like other land plants, cognate target genes corresponding to conserved miRNAs could be found in the genome database and were experimentally confirmed to guide cleavage of target mRNAs. The results suggested that two genes in the SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factor family, which are regulated by miR156 in most land plants, were instead targeted by two distinct miRNAs in M. polymorpha. In order to demonstrate the physiological roles of miRNAs in M. polymorpha, we constructed an miRNA ectopic expression system to establish overexpression transformants for conserved miRNAs, miR166 and miR319. Ectopic expression of these miRNAs induced abnormal development of the thallus and gemma cups, suggesting that balanced expression of miRNA/target mRNAs has a crucial role in developmental regulation in M. polymorpha. Profiling data on miRNA together with the ectopic expression system would provide new information on the liverwort small RNA world and evolutionary divergence/conservation of small RNA function among land plants.