miR156 Overexpression Research Articles

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.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Gibberellin and the miRNA156-targeted SlSBPs 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 (GA) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, we show GA and miR156-targeted SlSBPs interact in tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low levels of miR156-silenced SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL/SBP), 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 miR156-resistant SlSBP15 allele (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 SlSBPs, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal 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.

Quantitative proteomic analysis of the role of miRNA156 in alfalfa under drought stress

Alfalfa is the most produced perennial forage crop in Canada. Drought stress is a major form of abiotic stress, affecting its productivity and annual yield. A small RNA, miR156, plays a major role in drought tolerance by downregulating downstream SPL genes, but its effects at the proteome level are unknown. In this study, the protein level perturbations of miR156 overexpression (A8) and empty vector (EV) control genotypes were compared under drought stress. Using label-free quantification, 3000 protein groups were identified, of which 68 were upregulated in A8 and 84 were downregulated relative to EV under control conditions. Conversely, under drought stress, 610 proteins were upregulated and only 52 proteins were downregulated in A8 relative to EV. Functional analysis using PlantRegMap showed that the enriched proteins are likely involved in biological and molecular processes including antioxidant response, response to stress, signal transduction and biosynthesis of secondary metabolites. These proteins/pathways might be involved in the enhancement of drought stress tolerance mediated by miR156. Protein groups related to signaling, such as MAP kinase, calcium-dependent protein kinase, protein phosphatase 2 C, and transcriptional regulators including bZIP and zinc finger proteins were found to be differentially expressed when a search was conducted against a drought stress gene database. The proteomic dataset was validated by immunoblotting of selected proteins. The results of this study provide a better understanding and insight into the role of miR156 in drought stress tolerance in alfalfa at the proteomic level.

Comparative Transcriptome Analysis Reveals the Effect of miR156a Overexpression on Mineral Nutrient Homeostasis in Nicotiana tabacum.

Mineral nutrition plays an important role in crop growth, yield and quality. MiR156 is a regulatory hub for growth and development. To date, the understanding of miR156-mediated mineral homeostasis is limited. In this study, we overexpressed Nta-miR156a in the tobacco cultivar TN90 and analyzed the effects of miR156 on mineral element homeostasis in tobacco by comparative transcriptome analysis. The results showed that the overexpression of miR156a caused significant morphological changes in transgenic tobacco. Chlorophyll and three anti-resistance markers, proline, total phenolics, and total flavonoids, were altered due to increased miR156 expression levels. Interestingly, the distribution of Cu, Mn, Zn, and Fe in different tissues of transgenic tobacco was disordered compared with that of the wild type. Comparative transcriptome analysis showed that the overexpression of miR156 resulted in 2656 significantly differentially expressed genes. The expression levels of several metal-transport-related genes, such as NtABC, NtZIP, NtHMA, and NtCAX, were significantly increased or decreased in transgenic tobacco. These results suggest that miR156 plays an essential role in regulating mineral homeostasis. Our study provides a new perspective for the further study of mineral nutrient homeostasis in plants.

Deciphering the regulatory network of miR156 in plant architecture and abiotic stress resistance of alfalfa (Medicago sativa) by transcriptome sequencing

To improve alfalfa (Medicago sativa) biomass yield and abiotic stress resistance is a challenge due to its autotetraploidy and self-incompatibility. Previously we have generated transgenic alfalfa lines overexpressing miR156 and showing excellent plant architecture. In this study, we investigated the regulation network of miR156 in these transgenic alfalfa lines. Based on 83,202 non-redundant full-length transcripts in a pool of roots, stems, leaves, flowers, pods, and apical buds, we identified 154 candidate genes as potential downstream targets of miR156, especially for M. sativa SQUAMOSA promoter binding protein-like genes (MsSPLs), and then identified 26 MsSPL gene family members in alfalfa genome. The expressions of MsSPL5B and MsSPL3 were downregulated in root and leaf respectively, whereas that of MsSPL4 was significantly downregulated in stem, leaf, root, and apical bud tissues due to miR156 overexpression. We revealed the key role of MsSPL4-mediated regulatory pathways in the substantial correlations between agronomic traits and gene expression, and further found several involved hub genes showing significant correlation with MsSPL4. In addition, MsSPL4 overexpression in tobacco enhanced salinity tolerance but inhibited bud induction. Our results provide strong evidence for understanding the roles of miR156 and MsSPLs in regulating plant architecture and abiotic stress responses in alfalfa.

OsSPL14 acts upstream of OsPIN1b and PILS6b to modulate axillary bud outgrowth by fine-tuning auxin transport in rice.

As a multigenic trait, rice tillering can optimize plant architecture for the maximum agronomic yield. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE14 (OsSPL14) has been demonstrated to be necessary and sufficient to inhibit rice branching, but the underlying mechanism remains largely unclear. Here, we demonstrated that OsSPL14, which is cleaved by miR529 and miR156, inhibits tillering by fine-tuning auxin transport in rice. RNA interference of OsSPL14 or miR529 and miR156 overexpression significantly increased the tiller number, whereas OsSPL14 overexpression decreased the tiller number. Histological analysis revealed that the OsSPL14-overexpressing line had normal initiation of axillary buds but inhibited outgrowth of tillers. Moreover, OsSPL14 was found to be responsive to indole-acetic acid and 1-naphthylphthalamic acid, and RNA interference of OsSPL14 reduced polar auxin transport and increased 1-naphthylphthalamic acid sensitivity of rice plants. Further analysis revealed that OsSPL14 directly binds to the promoter of PIN-FORMED 1b (OsPIN1b) and PIN-LIKE6b (PILS6b) to regulate their expression positively. OsPIN1b and PILS6b were highly expressed in axillary buds and proved involved in bud outgrowth. Loss of function of OsPIN1b or PILS6b increased the tiller number of rice. Taken together, our findings suggested that OsSPL14 could control axillary bud outgrowth and tiller number by activating the expression of OsPIN1b and PILS6b to fine-tune auxin transport in rice.

Flowering time regulation by the miRNA156 in the beet (Beta vulgaris ssp. maritima)

Successful reproduction of the plants is considered as a critical challenge in meeting the nutritional needs of the growing world population. A major significant change in the plant's life cycle is the transition from vegetative to reproductive phase. The flowering and bolting processes are influenced by several genetic and physiological factors. Molecular and genetic studies have identified key factors in age-dependent gene regulatory pathways such as miR172-miR156-SPLs module in phase transition in plants. Unlike in model plants, the role of miR172-miR156-SPLs like regulatory networks remains unexplored in crops such as Beta vulgaris. In the present study, for the first time, miR156 from Beta vulgaris was identified and characterized. miR156 gene from Beta vulgaris was cloned and ectopically overexpressed under the control of strong 35S promoter in sugar beet explants. Subsequently, transcript levels of the miR156 and its known targets (SPL4 and SPL9) were quantified in the transgenic and control plants. Consistent with reported role of mir156 in downregulation of SPL4 and SPL9, miR156 overexpression plants exhibited delayed flowering and atypical rooting pattern indicative of abnormal root development and poor root foraging performance. Altogether, our results suggest that Beta vulgaris miR156 modulate phase transition by acting as negative regulator of SPL4 and SPL9.

Hierarchical Action of Mulberry miR156 in the Vegetative Phase Transition.

The vegetative phase transition is a prerequisite for flowering in angiosperm plants. Mulberry miR156 has been confirmed to be a crucial factor in the vegetative phase transition in Arabidopsis thaliana. The over-expression of miR156 in transgenic Populus × canadensis dramatically prolongs the juvenile phase. Here, we find that the expression of mno-miR156 decreases with age in all tissues in mulberry, which led us to study the hierarchical action of miR156 in mulberry. Utilizing degradome sequencing and dual-luciferase reporter assays, nine MnSPLs were shown to be directly regulated by miR156. The results of yeast one-hybrid and dual-luciferase reporter assays also revealed that six MnSPLs could recognize the promoter sequences of mno-miR172 and activate its expression. Our results demonstrate that mno-miR156 performs its role by repressing MnSPL/mno-miR172 pathway expression in mulberry. This work uncovered a miR156/SPLs/miR172 regulation pathway in the development of mulberry and fills a gap in our knowledge about the molecular mechanism of vegetative phase transition in perennial woody plants.

Heterogeneous expression of Osa-MIR156bc increases abiotic stress resistance and forage quality of alfalfa

Alfalfa (Medicago sativa L.) is the most widely cultivated perennial leguminous forage crop woldwide. MicroRNA156 (miR156) precursor genes from dicotyledonous species are reportedly useful for improving alfalfa plant architecture and abiotic stress resistance. However, there has been no report on whether a miR156 precursor gene from a monocotyledonous species functions in alfalfa. We introduced two tandem precursor genes of miR156, rice Osa-MIR156b and Osa-MIR156c (Osa-MIR156bc), into alfalfa. The expression of miR156 in the transgenic (TG) alfalfa was significantly elevated. Compared to wild-type plants, the TG plants overexpressing miR156 had more branches and leaves and showed improved salt and drought tolerance. Overexpression of miR156 slightly reduced plant height, but the biomass yield of TG plants grown in flowerpots was still increased. Forage quality of TG plants was markedly improved by reduction of acid detergent lignin (ADL) content and increase in crude protein content. The expression of the putative miR156 target genes MsSPL6, MsSPL12, and MsSPL13 in TG plants was repressed by miR156 overexpression, and that of all tested MsSPLs would be sharply increased under drought or salt stress. RNA sequencing revealed that overexpression of miR156 affected the expression of genes associated with abiotic stress resistance and plant development in multiple pathways. This first report of overexpression of monocot miR156 precursors in alfalfa sheds light on the function of miRNA156 precursors from the monocot species rice that could be used for genetic improvement of the dicot forage crop alfalfa.

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.

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.

A novel strigolactone-miR156 module controls stomatal behaviour during drought recovery.

miR156 is a conserved microRNA whose role and induction mechanisms under stress are poorly known. Strigolactones are phytohormones needed in shoots for drought acclimation. They promote stomatal closure ABA-dependently and independently; however, downstream effectors for the former have not been identified. Linkage between miR156 and strigolactones under stress has not been reported. We compared ABA accumulation and sensitivity as well as performances of wt and miR156-overexpressing (miR156-oe) tomato plants during drought. We also quantified miR156 levels in wt, strigolactone-depleted and strigolactone-treated plants, exposed to drought stress. Under irrigated conditions, miR156 overexpression and strigolactone treatment led to lower stomatal conductance and higher ABA sensitivity. Exogenous strigolactones were sufficient for miR156 accumulation in leaves, while endogenous strigolactones were required for miR156 induction by drought. The "after-effect" of drought, by which stomata do not completely re-open after rewatering, was enhanced by both strigolactones and miR156. The transcript profiles of several miR156 targets were altered in strigolactone-depleted plants. Our results show that strigolactones act as a molecular link between drought and miR156 in tomato, and identify miR156 as a mediator of ABA-dependent effect of strigolactones on the after-effect of drought on stomata. Thus, we provide insights into both strigolactone and miR156 action on stomata.

The interplay between miR156/SPL13 and DFR/WD40\u20131 regulate drought tolerance in alfalfa

BackgroundDeveloping Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop’s sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors.ResultsTo understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40–1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40–1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40–1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression.ConclusionsTaken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40–1 expression, whereas higher miR156 overexpression results in drought susceptibility.

PcG Proteins MSI1 and BMI1 Function Upstream of miR156 to Regulate Aerial Tuber Formation in Potato.

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member, represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon, whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects, including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhanced miR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further, grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increased accumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover, we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax group H3K4-methyl-transferases in StMSI1-OE Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.

MiR156/SPL9 Regulates Reactive Oxygen Species Accumulation and Immune Response in Arabidopsis thaliana.

The functions of microRNA156 (miR156) and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor genes in plant development have been widely investigated. However, the role of the miR156/SPLs regulatory network in plant immune systems remains obscure. Here, we found that the accumulation of reactive oxygen species (ROS) and the transcripts of basal salicylic acid (SA) signaling pathway genes were lower in Arabidopsis Pro35S:MIR156 seedlings (miR156 overexpression mutants) but higher in Pro35S:MIM156 (miR156 repression mutants) and ProSPL9:rSPL9 (SPL9 overexpression mutants) seedlings compared with wild-type Col-0 plants (WT). As a result, Pro35S:MIR156 mutants induced greater susceptibility to Pseudomonas syringae pv. tomato DC3000 following syringe infiltration than WT, while Pro35S:MIM156 and ProSPL9:rSPL9 mutants showed enhanced resistance. In addition, foliar H2O2 application resulted in activation of SA-mediated defense response and ablation of miR156-induced susceptibility to P. syringae pv. tomato DC3000 infection. Collectively, our results provide new insights into the function of the miR156/SPL network in Arabidopsis immune response by regulating ROS accumulation and activating the SA signaling pathway.

PSI-22 Effects of miR156 overexpression and silencing of SPL6 and SPL13 genes on the carbohydrate molecular structures in alfalfa.

The objectives of this study were to investigate the effect of overexpression of miR156 (miR156 OE) on carbohydrate molecular structures of alfalfa, in comparison with silencing SPL6 and SPL13 genes (SPL RNAi). Five miR156 OE, three SPL6-silenced (SPL6 RNAi) and three SPL13-silenced (SPL13 RNAi) alfalfa plants were grown with wild type (WT) under greenhouse conditions. Three batches of each genotype were harvested at mid-vegetative stage, freeze-dried and ground through 0.02mm sieve. Spectra were collected with attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Univariate analysis was performed with OMNIC software and spectral parameters were analyzed with PROC MIXED from SAS 9.4. Hierarchical cluster analysis (HCA) and principle component analysis (PCA) were performed with R software using both original spectra and second derivatives. Results showed that miR156 OE and SPL6/13 RNAi had lower heights and areas of total carbohydrate (TC), cellulosic compounds (CEC) and structural carbohydrate (STC) compared to WT, except that both WT and miR156 OE had higher TC4 and STC4 heights than SPL RNAi. Moreover, miR156 OE had greater STC2 and STC3 heights, and STC area (STCA) compared to SPL6/13 RNAi, and lower CEC height than SPL13 RNAi. As for PCA results, WT could be separated from SPL RNAi in CEC and STC regions with original spectra, and in STC and total carbohydrate (CHO) regions by using second derivatives. In terms of HCA, original spectra clusters distinguished WT from SPL RNAi in TC and STC regions. In contrast, second derivatives clusters could separate WT from both SPL RNAi and miR156 OE in STC and CHO regions. In conclusion, overexpression of miR156 altered the carbohydrate structures of alfalfa, which was similar as silencing of SPL6 and SPL13 genes. The STC region had the most spectral variations in carbohydrate region and could be used for clustering analysis.

Alfalfa response to heat stress is modulated by microRNA156.

Heat stress and extreme temperatures negatively affect plant development by disrupting regular cellular and biochemical functions, ultimately leading to reduced crop production. Alfalfa (Medicago sativa) is an important forage crop grown worldwide as forage for livestock feed. Limiting the effects of abiotic stress by developing alfalfa cultivars that are stress tolerant would help mitigate losses to crop production. Members of the microRNA156 (miR156) family regulate the Squamosa Promoter-Binding Protein-Like (SPL) genes that in turn impact plant growth and development by regulating downstream genes in response to various abiotic stresses. In this study, alfalfa with miR156 overexpression and SPL13 RNAi knockdown show increased tolerance to heat stress (40°C). Transgenic plants show high water potential and increased non-enzymatic antioxidant content under heat stress. Moreover, anthocyanin content and chlorophyll abundance were increased under stress. Expression of some important transcription factors and downstream genes involved in abiotic stress response were altered in miR156-overexpressing genotypes under heat. Taken together, our results demonstrate that the miR156/SPL13 network contributes to improving heat stress tolerance in alfalfa.

MiR156-SPL modules regulate induction of somatic embryogenesis in citrus callus.

miR156 is a highly conserved plant miRNA and has been extensively studied because of its versatile roles in plant development. Here, we report a novel role of miR156 in regulating somatic embryogenesis (SE) in citrus, one of the most widely cultivated fruit crops in the world. SE is an important means of in vitro regeneration, but over the course of long-term sub-culturing there is always a decline in the SE potential of the preserved citrus embryogenic callus, and this represents a key obstacle for citrus biotechnology. In this study, the SE competence of citrus callus of wild kumquat (Fortunella hindsii) was significantly enhanced by either overexpression of csi-miR156a or by individual knock-down of the two target genes, CsSPL3 and CsSPL14, indicating that the effect of miR156-SPL modules was established during the initial phases of SE induction. Biological processes that might promote SE in response to miR156 overexpression were explored using RNA-seq, and mainly included hormone signaling pathways, stress responses, DNA methylation, and the cell cycle. CsAKIN10 was identified as interacting protein of CsSPL14. Our results provide insights into the regulatory pathway through which miR156-SPL modules enhance the SE potential of citrus callus, and provide a theoretical basis for improvement of plant SE competence.

MiR156/SPL10 Modulates Lateral Root Development, Branching and Leaf Morphology in Arabidopsis by Silencing AGAMOUS-LIKE 79.

The developmental functions of miR156-SPL regulatory network have been extensively studied in Arabidopsis, but the downstream genes regulated by each SPL have not been well characterized. In this study, Next Generation Sequencing-based transcriptome analysis was performed on roots of wild type (WT) and miR156 overexpression (miR156OE) plants. One of the SPL genes, SPL10, which represses lateral root growth in Arabidopsis, was significantly downregulated in miR156OE plants. A transcription factor, AGAMOUS-like MADS box protein 79 (AGL79), was also significantly downregulated in the miR156OE plants, but was upregulated in the SPL10 overexpression (SPL10OE) Arabidopsis plants. In addition, SPL10 was found to bind to the core consensus SPL binding sequences in AGL79 gene. Moreover, analyses of complementation lines revealed a linear relationship between SPL10 and AGL79 in regulating Arabidopsis plant development. In addition, it was observed that plant phenotypes are AGL79 dose-dependent, with higher expression causing narrow leaf shape, less number of leaves and early flowering time, whereas relatively lower AGL79 overexpression produce plants with more rosette leaves and more lateral branches. Our findings revealed direct binding of SPL10 to AGL79 promoter, which further suggests a role for miR156/SPL10 module in plant lateral root growth by directly regulating AGL79.