Gibberellin and miRNA156-targeted SlSBP genes synergistically regulate tomato floral meristem determinacy and ovary patterning.

Many developmental processes associated with fruit development occur at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GAs) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, in tomato (Solanum lycopersicum), we show that GA and miR156-targeted SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL or SBP) genes interact in the tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low expression levels of miR156-silenced SBP genes, resulted in enlarged FMs, ovary indeterminacy and fruits with increased locule number. Conversely, low GA responses reduced indeterminacy and locule number, and overexpression of a S. lycopersicum (Sl)SBP15 allele that is miR156 resistant (rSBP15) reduced FM size and locule number. GA responses were partially required for the defects observed in 156OE and rSBP15 fruits. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBP genes, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to fine-tune aspects of fruit development that have been important for tomato domestication.

miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

The genetic basis of natural variation in the timing of vegetative phase change in Arabidopsis thaliana.

The juvenile-to-adult transition in plants is known as vegetative phase change and is marked by changes in the expression of leaf traits in response to a decrease in the level of miR156 and miR157. To determine whether this is the only mechanism of vegetative phase change, we measured the appearance of phase-specific leaf traits in 70 natural accessions of Arabidopsis thaliana. We found that leaf shape was poorly correlated with abaxial trichome production (two adult traits), that variation in these traits was not necessarily correlated with the level of miR156, and that there was little to no correlation between the appearance of adult-specific vegetative traits and flowering time. We identified eight quantitative trait loci controlling phase-specific vegetative traits from a cross between the Columbia (Col-0) and Shakdara (Sha) accessions. Only one of these quantitative trait loci includes genes known to regulate vegetative phase change (MIR156A and TOE1), which were expressed at levels consistent with the precocious phenotype of Sha. Our results suggest that vegetative phase change is regulated both by the miR156/SPL module and by genes specific to different vegetative traits, and that natural variation in vegetative phase change can arise from either source.

Screening of mtr-miR156a from exosomes of dairy cow blood to milk and its regulatory effect on milk protein synthesis in BMECs.

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows.

SPL16 and SPL23 mediate photoperiodic control of seasonal growth in Populus trees.

Perennial trees in boreal and temperate regions undergo growth cessation and bud set under short photoperiods, which are regulated by phytochrome B (phyB) photoreceptors and PHYTOCHROME INTERACTING FACTOR 8 (PIF8) proteins. However, the direct signaling components downstream of the phyB-PIF8 module remain unclear. We found that short photoperiods suppressed the expression of miR156, while upregulated the expression of miR156-targeted SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 16 (SPL16) and SPL23 in leaves and shoot apices of Populus trees. Accordingly, either overexpression of MIR156a/c or mutagenesis of SPL16/23 resulted in the attenuation of growth cessation and bud set under short days (SD), whereas overexpression of SPL16 and SPL23 conferred early growth cessation. We further showed that SPL16 and SPL23 directly suppressed FLOWERING LOCUS T2 (FT2) expression while promoted BRANCHED1 (BRC1.1 and BRC1.2) expression. Moreover, we revealed that PIF8.1/8.2, positive regulators of growth cessation, directly bound to promoters of MIR156a and MIR156c and inhibited their expression to modulate downstream pathways. Our results reveal a connection between the phyB-PIF8 module-mediated photoperiod perception and the miR156-SPL16/23-FT2/BRC1 regulatory cascades in SD-induced growth cessation. Our study provides insights into the rewiring of a conserved miR156-SPL module in the regulation of seasonal growth in Populus trees.

Cell division in the shoot apical meristem is a trigger for miR156 decline and vegetative phase transition in Arabidopsis.

What determines the rate at which a multicellular organism matures is a fundamental question in biology. In plants, the decline of miR156 with age serves as an intrinsic, evolutionarily conserved timer for the juvenile-to-adult phase transition. However, the way in which age regulates miR156 abundance is poorly understood. Here, we show that the rate of decline in miR156 is correlated with developmental age rather than chronological age. Mechanistically, we found that cell division in the apical meristem is a trigger for miR156 decline. The transcriptional activity of MIR156 genes is gradually attenuated by the deposition of the repressive histone mark H3K27me3 along with cell division. Our findings thus provide a plausible explanation of why the maturation program of a multicellular organism is unidirectional and irreversible under normal growth conditions and suggest that cell quiescence is the fountain of youth in plants.

miR156-SPL and miR169-NF-YA Modules Regulate the Induction of Somatic Embryogenesis in Arabidopsis via LEC- and Auxin-Related Pathways.

The embryogenic transition of plant somatic cells to produce somatic embryos requires extensive reprogramming of the cell transcriptome. The prominent role of transcription factors (TFs) and miRNAs in controlling somatic embryogenesis (SE) induction in plants was documented. The profiling of MIRNA expression in the embryogenic culture of Arabidopsis implied the contribution of the miR156 and miR169 to the embryogenic induction. In the present study, the function of miR156 and miR169 and the candidate targets, SPL and NF-YA genes, were investigated in Arabidopsis SE. The results showed that misexpression of MIRNA156 and candidate SPL target genes (SPL2, 3, 4, 5, 9, 10, 11, 13, 15) negatively affected the embryogenic potential of transgenic explants, suggesting that specific fine-tuning of the miR156 and target genes expression levels seems essential for efficient SE induction. The results revealed that SPL11 under the control of miR156 might contribute to SE induction by regulating the master regulators of SE, the LEC (LEAFY COTYLEDON) genes (LEC1, LEC2, FUS3). Moreover, the role of miR169 and its candidate NF-YA targets in SE induction was demonstrated. The results showed that several miR169 targets, including NF-YA1, 3, 5, 8, and 10, positively regulated SE. We found, that miR169 via NF-YA5 seems to modulate the expression of a master SE regulator LEC1/NF-YA and other auxin-related genes: YUCCA (YUC4, 10) and PIN1 in SE induction. The study provided new insights into miR156-SPL and miR169-NF-YA functions in the auxin-related and LEC-controlled regulatory network of SE.

MicroRNAs Participate in Morphological Acclimation of Sugar Beet Roots to Nitrogen Deficiency.

Nitrogen (N) is essential for sugar beet (Beta vulgaris L.), a highly N-demanding sugar crop. This study investigated the morphological, subcellular, and microRNA-regulated responses of sugar beet roots to low N (LN) stress (0.5 mmol/L N) to better understand the N perception, uptake, and utilization in this species. The results showed that LN led to decreased dry weight of roots, N accumulation, and N dry matter production efficiency, along with damage to cell walls and membranes and a reduction in organelle numbers (particularly mitochondria). Meanwhile, there was an increase in root length (7.2%) and branch numbers (29.2%) and a decrease in root surface area (6.14%) and root volume (6.23%) in sugar beet after 7 d of LN exposure compared to the control (5 mmol/L N). Transcriptomics analysis was confirmed by qRT-PCR for 6 randomly selected microRNAs, and we identified 22 differentially expressed microRNAs (DEMs) in beet root under LN treatment. They were primarily enriched in functions related to binding (1125), ion binding (641), intracellular (437) and intracellular parts (428), and organelles (350) and associated with starch and sucrose metabolism, tyrosine metabolism, pyrimidine metabolism, amino sugar and nucleotide sugar metabolism, and isoquinoline alkaloid biosynthesis, as indicated by the GO and KEGG analyses. Among them, the upregulated miR156a, with conserved sequences, was identified as a key DEM that potentially targets and regulates squamosa promoter-binding-like proteins (SPLs, 104889216 and 104897537) through the microRNA-mRNA network. Overexpression of miR156a (MIR) promoted root growth in transgenic Arabidopsis, increasing the length, surface area, and volume. In contrast, silencing miR156a (STTM) had the opposite effect. Notably, the fresh root weight decreased by 45.6% in STTM lines, while it increased by 27.4% in MIR lines, compared to the wild type (WT). It can be inferred that microRNAs, especially miR156, play crucial roles in sugar beet root's development and acclimation to LN conditions. They likely facilitate active responses to N deficiency through network regulation, enabling beet roots to take up nutrients from the environment and sustain their vital life processes.

Flowering onset time is regulated by microRNA-mediated trehalose-6-phosphate signaling in Cajanus cajan L. under elevated CO2.

CO2 levels are known to have an impact on plant development and physiology. In the current study, we have investigated the effect of elevated CO2 on flowering and its regulation through miRNA mediated sugar signaling. We also unraveled small RNA transcriptome of pigeonpea under ambient and elevated CO2 conditions and predicted the targets for crucial miRNAs through computational methods. The results have shown that the delayed flowering in pigeonpea under elevated CO2 was due to an imbalance in C:N stoichiometry and differential expression pattern of aging pathway genes, including SQUAMOSA PROMOTER BINDING PROTEIN-LIKE. Furthermore, qRT PCR analysis has revealed the role of miR156 and miR172 in mediating trehalose-6-phosphate dependent flowering regulation. The current study is crucial in understanding the responses of flowering patterns in a legume crop to elevated CO2 which showed a significant impact on its final yields. Also, these findings are crucial in devising effective crop improvement strategies for developing climate resilient crops, including pigeonpea. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01434-9.

ALTERED MERISTEM PROGRAM1 regulates leaf identity independently of miR156-mediated translational repression.

In Arabidopsis, loss of the carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 might promote SPL activity. However, we found that the amp1 mutant phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although AMP1 has been reported to promote miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target SPL9 or the miR159 target MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway, and that it is not universally required for miRNA-mediated translational repression.

Screening of microRNAs and target genes involved in Sclerotinia sclerotiorum (Lib.) infection in Brassica napus L.

BACKGROUND: Rapeseed (Brassica napus L.) is the third largest source of vegetable oil in the world, and Sclerotinia sclerotiorum (Lib.) is a major soil-borne fungal plant pathogen that infects more than 400 plant species, including B. napus. Sclerotinia stem rot caused an annual loss of 10 - 20% in rapeseed yield. Exploring the molecular mechanisms in response to S. sclerotiorum infection in B. napus is beneficial for breeding and cultivation of resistant varieties. To gain a better understanding of the mechanisms regarding B. napus tolerance to Sclerotinia stem rot, we employed a miRNAome sequencing approach and comprehensively investigated global miRNA expression profile among five relatively resistant lines and five susceptible lines of oilseed at 0, 24, and 48 h post-inoculation. RESULTS: In this study, a total of 40 known and 1105 novel miRNAs were differentially expressed after S. sclerotiorum infection, including miR156, miR6028, miR394, miR390, miR395, miR166, miR171, miR167, miR164, and miR172. Furthermore, 8,523 genes were predicted as targets for these differentially expressed miRNAs. These target genes were mainly associated with disease resistance (R) genes, signal transduction, transcription factors, and hormones. Constitutively expressing miR156b (OX156b) plants strengthened Arabidopsis resistance against S. sclerotiorum accompanied by smaller necrotic lesions, whereas blocking miR156 expression in Arabidopsis (MIM156) led to greater susceptibility to S. sclerotiorum disease, associated with extensive cell death of necrotic lesions. CONCLUSIONS: This study reveals the distinct difference in miRNA profiling between the relatively resistant lines and susceptible lines of B. napus in response to S. sclerotiorum. The identified differentially expressed miRNAs related to sclerotinia stem rot resistance are involved in regulating resistance to S. sclerotiorum in rapeseed by targeting genes related to R genes, signal transduction, transcription factors, and hormones. miR156 positively modulates the resistance to S. sclerotiorum infection by restricting colonization of S. sclerotiorum mycelia. This study provides a broad view of miRNA expression changes after S. sclerotiorum infection in oilseed and is the first to elucidate the function and mechanism underlying the miR156 response to S. sclerotiorum infection in oilseed rape.

The miR156b-GmSPL2b module mediates male fertility regulation of cytoplasmic male sterility-based restorer line under high-temperature stress in soybean.

High-temperature (HT) stress at flowering stage causes significant damage to soybean, including pollen abortion and fertilization failure, but few genes involved in male fertility regulation under HT stress in soybean have been characterized. Here, we demonstrated that miR156b-GmSPL2b module involved in male fertility regulation of soybean cytoplasmic male sterility (CMS)-based restorer line under HT stress. Overexpression of miR156b decreased male fertility in soybean CMS-based restorer line and its hybrid F1 with CMS line under HT stress. RNA-seq analysis found that miR156b mediated male fertility regulation in soybean under HT stress by regulating the expression of pollen development and HT response related genes. Metabolomic analysis of miR156bOE revealed reduction in flavonoid content under HT stress. Integrated transcriptomic and metabolomic analysis showed that the overexpression of miR156b caused flavonoid metabolism disorder in soybean flower bud under HT stress. Knockout of GmSPL2b also decreased the thermotolerance of soybean CMS-based restorer line during flowering. Moreover, GmSPL2b turned out to be directly bounded to the promoter of GmHSFA6b. Further verification indicated that GmHSFA6b overexpression enhanced HT tolerance in Arabidopsis during flowering. Substance content and gene expression analysis revealed that miR156b-GmSPL2b may mediate reactive oxygen species clearance by regulating flavonoid metabolism, thus participating in the regulation of male fertility in soybean under HT stress. This study not only provided important progress for understanding the molecular mechanism of miR156b-GmSPL2b regulating the male fertility of soybean CMS-based restorer line under HT stress, but also provided genetic resources and theoretical basis for creating HT-tolerant strong restorer lines.

Tomato miR156-targeted SlSBP15 represses shoot branching by modulating hormone dynamics and interacting with GOBLET and BRANCHED1b.

The miRNA156 (miR156)/SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL/SBP) regulatory hub is highly conserved among phylogenetically distinct species, but how it interconnects multiple pathways to converge to common integrators controlling shoot architecture is still unclear. Here, we demonstrated that the miR156/SlSBP15 node modulates tomato shoot branching by connecting multiple phytohormones with classical genetic pathways regulating both axillary bud development and outgrowth. miR156-overexpressing plants (156-OE) displayed high shoot branching, whereas plants overexpressing a miR156-resistant SlSBP15 allele (rSBP15) showed arrested shoot branching. Importantly, the rSBP15 allele was able to partially restore the wild-type shoot branching phenotype in the 156-OE background. rSBP15 plants have tiny axillary buds, and their activation is dependent on shoot apex-derived auxin transport inhibition. Hormonal measurements revealed that indole-3-acetic acid (IAA) and abscisic acid (ABA) concentrations were lower in 156-OE and higher in rSBP15 axillary buds, respectively. Genetic and molecular data indicated that SlSBP15 regulates axillary bud development and outgrowth by inhibiting auxin transport and GOBLET (GOB) activity, and by interacting with tomato BRANCHED1b (SlBRC1b) to control ABA levels within axillary buds. Collectively, our data provide a new mechanism by which the miR156/SPL/SBP hub regulates shoot branching, and suggest that modulating SlSBP15 activity might have potential applications in shaping tomato shoot architecture.

Genome-Wide Analysis of SPL/miR156 Module and Its Expression Analysis in Vegetative and Reproductive Organs of Oil Palm (Elaeis guineensis).

The SPL (SQUAMOSA-promoter binding protein-like) gene family is one of the largest plant transcription factors and is known to be involved in the regulation of plant growth, development, and stress responses. The genome-wide analysis of SPL gene members in a diverse range of crops has been elucidated. However, none of the genome-wide studies on the SPL gene family have been carried out for oil palm, an important oil-yielding plant. In this research, a total of 24 EgSPL genes were identified via a genome-wide approach. Phylogenetic analysis revealed that most of the EgSPLs are closely related to the Arabidopsis and rice SPL gene members. EgSPL genes were mapped onto the only nine chromosomes of the oil palm genome. Motif analysis revealed conservation of the SBP domain and the occurrence of 1-10 motifs in EgSPL gene members. Gene duplication analysis demonstrated the tandem duplication of SPL members in the oil palm genome. Heatmap analysis indicated the significant expression of SPL genes in shoot and flower organs of oil palm plants. Among the identified EgSPL genes, a total 14 EgSPLs were shown to be targets of miR156. Real-time PCR analysis of 14 SPL genes showed that most of the EgSPL genes were more highly expressed in female and male inflorescences of oil palm plants than in vegetative tissues. Altogether, the present study revealed the significant role of EgSPL genes in inflorescence development.

The MdmiR156n Regulates Drought Tolerance and Flavonoid Synthesis in Apple Calli and Arabidopsis.

Drought is the major abiotic stress that limits apple productivity and quality. To date, many important and divergent regulatory functions of miR156/SBP genes in plant growth and development have been well understood. However, little is known about the role of apple miR156 in response to abiotic stress. To better understand the functions of MdmiR156 in abiotic stress tolerance, we constructed the overexpression (OE) and short tandem target mimic (STTM) vector of MdmiR156n and performed its functional analysis through the characterization of transgenic apple calli and Arabidopsis thaliana plants. In this study, MdmiR156n overexpression significantly increased the length of primary roots and the number of lateral roots in transgenic Arabidopsis plants under drought stress. In addition, MdmiR156n transgenic Arabidopsis and apple calli had a lower electrolyte leakage rate and less cell membrane damage than WT and STTM156 after drought stress. Further studies showed that MdmiR156n overexpression promoted the accumulation of flavonoids and scavenging of reactive oxygen species (ROS) under drought conditions in transgenic apple calli and A. thaliana plants. Taken together, overexpression MdmiR156n enhances drought tolerance by regulating flavonoid synthesis and ROS signaling cascades in apple calli and A. thaliana.

The Competing Endogenous RNAs Regulatory Genes Network Mediates Leaf Shape Variation and Main Effector Gene Function in Mulberry Plant (Morus alba).

Mulberry plants (Morus alba) have leaf shapes, ranging from unlobed to lobed, which are crucial for yield, growth, and adaptability, indicating their ability to adapt to their environment. Competing endogenous RNAs (ceRNAs) constitute a web of RNAs within the organism's transcriptional regulatory system, including protein-coding genes (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and others. In this study, samples for ceRNA sequencing were categorized into two groups: whole leaves and lobed leaves, each group with three replicates. In addition, we isolated, cloned, and characterized the precursor miRNA (miR156x) from the leaves of M. alba. miR156x precursor had a length of 107 base pairs and a minimum folding free energy of 50.27 kcal/mol. We constructed a pCAMBIA-35S-GUS-miR156x dual overexpression vector and established a transient transformation system for mulberry. At an optimal transformation solution (OD600 = 0.7), the GUS gene showed a higher expression in the leaves of transiently transformed mulberry with miR156x overexpression, four days after transformation, while the target genes of miR156x had decreased expression in the same leaves. Investigations into the transgenic mulberry plants uncovered various modifications to physio-chemical parameters including POD, SOD, PRO, MDA, soluble proteins and sugars, and chlorophyl content. miRNAs in the plants were found to act as negative regulators of gene expression in response to changes in leaf shape regulation, which was confirmed in vitro using dual-luciferase reporter assays. Subsequently, we cloned Maspl3 in vitro and conducted GST-Pull down assays, obtaining multiple proteins that interacted with the Maspl3 gene. This indicates that the miR156x/Maspl3/MSTRG.25812.1 regulatory module contributes to the differences in mulberry leaf shape.

SQUINT Positively Regulates Resistance to the Pathogen Botrytis cinerea via miR156-SPL9 Module in Arabidopsis.

SQUINT (SQN) regulates plant maturation by promoting the activity of miR156, which functions primarily in the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) module regulating plant growth and development. Here, we show that SQN acts in the jasmonate (JA) pathway, a major signaling pathway regulating plant responses to insect herbivory and pathogen infection. Arabidopsis thaliana sqn mutants showed elevated sensitivity to the necrotrophic fungus Botrytis cinerea compared with wild type. However, SQN is not involved in the early pattern-triggered immunity response often triggered by fungal attack. Rather, SQN positively regulates the JA pathway, as sqn loss-of-function mutants treated with B. cinerea showed reduced JA accumulation, JA response and sensitivity to JA. Furthermore, the miR156-SPL9 module regulates plant resistance to B. cinerea: mir156 mutant, and SPL9 overexpression plants displayed elevated sensitivity to B. cinerea. Moreover, constitutively expressing miR156a or reducing SPL9 expression in the sqn-1 mutant restored the sensitivity of Arabidopsis to B. cinerea and JA responses. These results suggest that SQN positively modulates plant resistance to B. cinerea through the JA pathway, and the miR156-SPL9 module functions as a bridge between SQN and JA to mediate plant resistance to this pathogen.

Genome-wide analysis and molecular dissection of the SPL gene family in Fraxinus mandshurica.

BACKGROUND: SQUAMOSA promoter binding protein-like (SPL) is a unique family of transcription factors in plants, which is engaged in regulating plant growth and development, physiological and biochemical processes. Fraxinus mandshurica is an excellent timber species with a wide range of uses in northeastern China and enjoys a high reputation in the international market. SPL family analysis has been reported in some plants while SPL family analysis of Fraxinus mandshurica has not been reported. RESULTS: We used phylogeny, conserved motifs, gene structure, secondary structure prediction, miR156 binding sites, promoter cis elements and GO annotation to systematically analyze the FmSPLs family. This was followed by expression analysis by subcellular localization, expression patterns at various tissue sites, abiotic stress and hormone induction. Because FmSPL2 is highly expressed in flowers it was selected to describe the SPL gene family of Fraxinus mandshurica by ectopic expression. Among them, 10 FmSPL genes that were highly expressed at different loci were selected for expression analysis under abiotic stress (NaCl and Cold) and hormone induction (IAA and ABA). These 10 FmSPL genes showed corresponding trends in response to both abiotic stress and hormone induction. We showed that overexpression of FmSPL2 in transgenic Nicotiana tabacum L. resulted in taller plants, shorter root length, increased root number, rounded leaves, and earlier flowering time. CONCLUSIONS: We identified 36 SPL genes, which were classified into seven subfamilies based on sequence analysis. FmSPL2 was selected for subsequent heterologous expression by analysis of expression patterns in various tissues and under abiotic stress and hormone induction, and significant phenotypic changes were observed in the transgenic Nicotiana tabacum L. These results provide insight into the evolutionary origin and biological significance of plant SPL. The aim of this study was to lay the foundation for the genetic improvement of Fraxinus mandshurica and the subsequent functional analysis of FmSPL2.

LATERAL BRANCHING OXIDOREDUCTASE, one novel target gene of Squamosa Promoter Binding Protein-like 2, regulates tillering in switchgrass.

Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late-acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild-type and miR156 overexpressing (miR156OE ) switchgrass. Co-expression analysis, quantitative real-time polymerase chain reaction (qRT-PCR), transient dual luciferase assay, and chromatin immunoprecipitation-qPCR were all used to determine the activation of PvLBO by miR156-targeted Squamosa Promoter Binding Protein-like 2 (PvSPL2) in regulating tillering of switchgrass. PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2. We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156-SPL module in switchgrass.

PICKLE associates with histone deacetylase 9 to mediate vegetative phase change in Arabidopsis.

The juvenile-to-adult vegetative phase change in flowering plants is mediated by a decrease in miR156 levels. Downregulation of MIR156A/MIR156C, the two major sources of miR156, is accompanied by a decrease in acetylation of histone 3 lysine 27 (H3K27ac) and an increase in trimethylation of H3K27 (H3K27me3) at MIR156A/MIR156C in Arabidopsis. Here, we show that histone deacetylase 9 (HDA9) is recruited to MIR156A/MIR156C during the juvenile phase and associates with the CHD3 chromatin remodeler PICKLE (PKL) to erase H3K27ac at MIR156A/MIR156C. H2Aub and H3K27me3 become enriched at MIR156A/MIR156C, and the recruitment of Polycomb Repressive Complex 2 (PRC2) to MIR156A/MIR156C is partially dependent on the activities of PKL and HDA9. Our results suggest that PKL associates with histone deacetylases to erase H3K27ac and promote PRC1 and PRC2 activities to mediate vegetative phase change and maintain plants in the adult phase after the phase transition.