D-optimal Design Optimization of Solvent Mixture for Flavonoid Extraction from Phalaenopsis Leaves with Antioxidant Activity.

Aims: This study aimed to optimize the extraction of flavonoids and antioxidants from Phalaenopsis leaves by using solvent mixtures. Method: The total flavonoid content (TFC) and antioxidant activity were evaluated using the colorimetric method and ferric-reducing antioxidant power (FRAP), respectively. Maceration extracts from fresh leaves were used for the analysis. The study used the Design Expert 13.0 program to optimize the solvents (water, acetone, and methanol) and their combined ratio. Result: The results showed that 100% acetone was the best solvent for both responses, with a desirability value of 0.884, TFC of 0.434 mg QE/g fresh weight (FW) and FRAP of 713.53 µmol TE/g FW. Screening of the most potent Phalaenopsis genotypes for obtaining the most active leaf extract showed that P. amboinensis and P. pantherina were the best genotypes for TFC (0.786-0.797 mg QE/g FW) and FRAP activity (862.25-891.48 µmol TE/g FW). Conclusion: This study demonstrates an easy and useful way to obtain flavonoids and antioxidants from Phalaenopsis materials that can be used in the flower-based industry to make new functional ingredients.

TALE-based organellar genome editing and gene expression in plants.

KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

Arabidopsis AtMSRB5 functions as a salt-stress protector for both Arabidopsis and rice.

Salinity, drought and low temperature are major environmental factors that adversely affect crop productivity worldwide. In this study we adopted an activation tagging approach to identify salt tolerant mutants of Arabidopsis. Thousands of tagged Arabidopsis lines were screened to obtain several potential mutant lines resistant to 150 mM NaCl. Transcript analysis of a salt-stress tolerance 1 (sst1) mutant line indicated activation of AtMSRB5 and AtMSRB6 which encode methionine sulfoxide reductases. Overexpression of AtMSRB5 in Arabidopsis (B5OX) showed a similar salt tolerant phenotype. Furthermore, biochemical analysis indicated stability of the membrane protein, H+-ATPase 2 (AHA2) through regulation of Na+/K+ homeostasis which may be involved in a stress tolerance mechanism. Similarly, overexpression of AtMSRB5 in transgenic rice demonstrated a salt tolerant phenotype via the modulation of Na+/K+ homeostasis without a yield drag under salt and oxidative stress conditions.

Optimal culture conditions of Piriformospora indica for volatile compound release to promote effective plant growth.

Piriformospora indica, which is an endophytic fungus that grows on various media in the absence of a host, emits plant growth promoting volatile organic compounds (VOCs). Kaefer medium (KF) has been shown to be the most suitable medium for P. indica growth; however, different media may differentially affect fungal metabolism which may in turn influence the VOC profiles of P. indica. To date, how the VOCs emitted from P. indica cultured on different media affect plant growth has not been well characterized. Here, we show that poor nutrient medium (PNM) promoted the growth of P. indica more effectively than potato dextrose agar (PDA) or KF medium. By contrast, plant total biomass and root fresh weight were increased 1.8-fold and 2.1-fold, when co-cultivated with P. indica cultured on PDA medium in comparison with KF or PNM medium, respectively. Furthermore, sucrose in the plant culture medium downregulated the fold-induction ratio of the plant growth promoted by P. indica VOCs.

Ethylene Response Factor109 Attunes Immunity, Photosynthesis, and Iron Homeostasis in Arabidopsis Leaves.

Iron (Fe) is an essential micronutrient element for all organisms including plants. Chlorosis of young leaves is a common symptom of Fe deficiency, reducing the efficiency of photosynthesis, and, ultimately, crop yield. Previous research revealed strong responsiveness of the putative key transcription factor ERF109 to the Fe regime. To elucidate the possible role of ERF109 in leaf Fe homeostasis and photosynthesis, we subjected Arabidopsis thaliana erf109 knockout lines and Col-0 wild-type plants to transcriptome profiling via RNA-seq. The transcriptome profile of Fe-sufficient erf109 leaves showed a 71% overlap with Fe-deficient Col-0 plants. On the other hand, genes that were differentially expressed between Fe-deficient and Fe-sufficient Col-0 plants remained unchanged in erf109 plants under conditions of Fe deficiency. Mutations in ERF109 increased the expression of the clade Ib bHLH proteins bHLH38, bHLH39, bHLH101, the nicotianamine synthase NAS4, and the Fe storage gene FER1. Moreover, mutations in ERF109 led to significant down-regulation of defense genes, including CML37, WRKY40, ERF13, and EXO70B2. Leaves of erf109 exhibited increased Fe levels under both Fe-sufficient and Fe-deficient conditions. Reduced Fv/Fm and Soil Plant Analysis Development (SPAD) values in erf109 lines under Fe deficiency indicate curtailed ability of photosynthesis relative to the wild-type. Our findings suggest that ERF109 is a negative regulator of the leaf response to Fe deficiency. It further appears that the function of ERF109 in the Fe response is critical for regulating pathogen defense and photosynthetic efficiency. Taken together, our study reveals a novel function of ERF109 and provides a systematic perspective on the intertwining of the immunity regulatory network and cellular Fe homeostasis.

Piriformospora indica symbiosis improves water stress tolerance of rice through regulating stomata behavior and ROS scavenging systems.

Global water shortage seriously threatens rice growth especially in irrigated production areas. Association of plants with beneficial soil microbes is one strategy for plant adaption to environmental stresses. In this study, rice (Oryza sativa L.) plants were colonized by the beneficial root-colonizing endophytic fungus Piriformospora indica (P. indica). We demonstrate that grain yield were higher in P. indica-colonized rice plants compared to the uncolonized plants grown in soil. Moreover, P. indica effect on improving water stress tolerance in rice and its physiological mechanism were investigated in a hydroponic culture system. Polyethylene glycol (PEG) was applied to the culture solution to conduct the water stress condition. Water stress-induced leaf wilting and impairments in photosynthetic efficiency were diminished in P. indica-colonized plants. Furthermore, P. indica colonization promotes stomata closure and increases the leaf surface temperature under water stress. The malondialdehyde level (as an indicator for oxidative stress) was lower and the reduced to oxidized glutathione ratio was higher in P. indica-colonized and PEG-exposed rice plants compared to the uncolonized plants. Furthermore, the activities of the antioxidant enzymes catalase and glutathione reductase were up-regulated in inoculated rice seedlings under water stress. In conclusion, P. indica promotes rice performance under water stress by stomata closure and lower oxidative stress.

Altered Gene Regulatory Networks Are Associated With the Transition From C3 to Crassulacean Acid Metabolism in Erycina (Oncidiinae: Orchidaceae).

Crassulacean acid metabolism (CAM) photosynthesis is a modification of the core C3 photosynthetic pathway that improves the ability of plants to assimilate carbon in water-limited environments. CAM plants fix CO2 mostly at night, when transpiration rates are low. All of the CAM pathway genes exist in ancestral C3 species, but the timing and magnitude of expression are greatly altered between C3 and CAM species. Understanding these regulatory changes is key to elucidating the mechanism by which CAM evolved from C3. Here, we use two closely related species in the Orchidaceae, Erycina pusilla (CAM) and Erycina crista-galli (C3), to conduct comparative transcriptomic analyses across multiple time points. Clustering of genes with expression variation across the diel cycle revealed some canonical CAM pathway genes similarly expressed in both species, regardless of photosynthetic pathway. However, gene network construction indicated that 149 gene families had significant differences in network connectivity and were further explored for these functional enrichments. Genes involved in light sensing and ABA signaling were some of the most differently connected genes between the C3 and CAM Erycina species, in agreement with the contrasting diel patterns of stomatal conductance in C3 and CAM plants. Our results suggest changes to transcriptional cascades are important for the transition from C3 to CAM photosynthesis in Erycina.

Phalaenopsis flowering locus VE regulates floral organ maturation.

KEY MESSAGE: PaFVE is low ambient temperature-inducible and acts as a systemic regulator in the early stage of floral development in Phalaenopsis. Phalaenopsis aphrodite: subsp. formosana, a native orchid species of Taiwan, is an economically important ornamental crop that requires low ambient temperature for floral transition. Currently, limited genetic information about such orchid species hampers genetic manipulation for specific or improved floral traits, and the control of flowering time independent of temperature regulation. In this study, the sequence of the full-length of Phalaenopsis flowering locus VE (PaFVE) gene was determined. Spatial and temporal expression studies showed that mRNA transcripts of PaFVE were inducible by low ambient temperature, and high levels of expression occurred after spiking initiation and remained high throughout the early stage of floral development. Further investigation revealed that floral organ development was impeded in PaFVE-silenced P. aphrodite, but flowering time and floral organogenesis were not compromised. Analysis of the downstream flowering genes suggested that the delay in floral maturation is associated with a corresponding decrease in the expression of downstream flowering genes, PaSOC1, PaSOC1L and PaAGL24. The ectopic expression of PaFVE in Arabidopsis resulted in an accelerated flowering time, accompanied by an increase in the expression of AtSOC1, thus revealing the functional role of PaFVE as a floral regulator. Overall, our results demonstrate that PaFVE has evolutionarily diverged and conserved functions, and serves as a regulator of floral organ maturation in Phalaenopsis and a regulator of flowering time in Arabidopsis.

Genome-wide analysis of GDSL-type esterases/lipases in Arabidopsis.

KEY MESSAGE: In this present study, we introduce a fundamental framework and provide information regarding the possible roles of GDSL-type esterase/lipase gene family in Arabidopsis. GDSL-type esterases/lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity, regiospecificity, and stereoselectivity. In this study, we identified 105 GDSL-type esterase/lipase genes in Arabidopsis thaliana by conducting a comprehensive computational analysis. Expression studies indicated that GDSL-type lipase proteins showed varied expression patterns. Phylogenetic tree analysis indicated that AtGELP (Arabidopsis thaliana GDSL-type esterase/lipase protein) gene family was divided into four clades. The phylogenetic analysis, combined with protein motif architectures, and expression profiling were used to predict the roles AtGELP genes. To investigate the physical roles of the AtGELP gene family, we successfully screened 88 AtGELP T-DNA knockout lines for 54 AtGELP genes from 199 putative SALK T-DNA mutants. Transgenic plants of AtGELP genes were used to elucidate the phenotypic characteristics in various developmental stages or stress conditions. Our results suggest that the AtGELP genes have diverse physical functions such as affecting the germination rate and early growth of seedlings subjected to high concentrations of glucose, or being involved in biotic stress responses.

The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system.

Plant defensins (PDFs) are cysteine-rich peptides that have a range of biological functions, including defence against fungal pathogens. However, little is known about their role in defence against bacteria. In this study, we showed that the protein encoded by ARABIDOPSIS THALIANA PLANT DEFENSIN TYPE 1.1 (AtPDF1.1) is a secreted protein that can chelate apoplastic iron. Transcripts of AtPDF1.1 were induced in both systemic non-infected leaves of Arabidopsis thaliana plants and those infected with the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc). The expression levels of AtPDF1.1 with correct subcellular localization in transgenic A. thaliana plants were positively correlated with tolerance to Pcc, suggesting its involvement in the defence against this bacterium. Expression analysis of genes associated with iron homeostasis/deficiency and hormone signalling indicated that the increased sequestration of iron by apoplastic AtPDF1.1 overexpression perturbs iron homeostasis in leaves and consequently activates an iron-deficiency-mediated response in roots via the ethylene signalling pathway. This in turn triggers ethylene-mediated signalling in systemic leaves, which is involved in suppressing the infection of necrotrophic pathogens. These findings provide new insight into the key functions of plant defensins in limiting the infection by the necrotrophic bacterium Pcc via an iron-deficiency-mediated defence response.

Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla.

Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS-box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS-box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full-length genomic sequences of these MADS-box genes were obtained from BAC clones. Of these, 27 were MIKC-type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKC(c) genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS-box domain, EpMADS27-YFP was localized in the nucleus. This characterization of the orchid MADS-box family genes provides useful information for both orchid breeding and studies of flowering and evolution.

NDH expression marks major transitions in plant evolution and reveals coordinate intracellular gene loss.

BACKGROUND: Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. RESULTS: Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa. CONCLUSION: The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.

The location and translocation of ndh genes of chloroplast origin in the Orchidaceae family.

The NAD(P)H dehydrogenase complex is encoded by 11 ndh genes in plant chloroplast (cp) genomes. However, ndh genes are truncated or deleted in some autotrophic Epidendroideae orchid cp genomes. To determine the evolutionary timing of the gene deletions and the genomic locations of the various ndh genes in orchids, the cp genomes of Vanilla planifolia, Paphiopedilum armeniacum, Paphiopedilum niveum, Cypripedium formosanum, Habenaria longidenticulata, Goodyera fumata and Masdevallia picturata were sequenced; these genomes represent Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae subfamilies. Four orchid cp genome sequences were found to contain a complete set of ndh genes. In other genomes, ndh deletions did not correlate to known taxonomic or evolutionary relationships and deletions occurred independently after the orchid family split into different subfamilies. In orchids lacking cp encoded ndh genes, non cp localized ndh sequences were identified. In Erycina pusilla, at least 10 truncated ndh gene fragments were found transferred to the mitochondrial (mt) genome. The phenomenon of orchid ndh transfer to the mt genome existed in ndh-deleted orchids and also in ndh containing species.

Arabidopsis SFAR4 is a novel GDSL-type esterase involved in fatty acid degradation and glucose tolerance.

BACKGROUND: SFARs (seed fatty acid reducers) belonging to the GDSL lipases/esterases family have been reported to reduce fatty acid storage and composition in mature Arabidopsis seeds. GDSL lipases/esterases are hydrolytic enzymes that possess multifunctional properties, such as broad substrate specificity, regiospecificity, and stereoselectivity. Studies on the physiological functions and biochemical characteristics of GDSL lipases/esterases in plants are limited, so it is important to elucidate the molecular functions of GDSL-type genes. RESULTS: We found that SFAR4 (At3g48460), a fatty acid reducer belonging to the Arabidopsis GDSL lipases/esterases family, was intensely expressed in embryo protrusion, early seedlings, and pollen. The characterization of recombinant SFAR4 protein indicated that it has short-length p-nitrophenyl esterase activity. In addition, SFAR4 enhanced the expression of genes involved in fatty acid metabolism during seed germination and seedling development. SFAR4 elevated the expression of COMATOSE, which transports fatty acids into peroxisomes, and of LACS6 and LACS7, which deliver long-chain acetyl-CoA for ß-oxidation. Furthermore, SFAR4 increased the transcription of PED1 and PNC1, which function in importing peroxisomal ATP required for fatty acid degradation. SFAR4 has another function on tolerance to high glucose concentrations but had no significant effects on the expression of the glucose sensor HXK1. CONCLUSIONS: The results demonstrated that SFAR4 is a GDSL-type esterase involved in fatty acid metabolism during post-germination and seedling development in Arabidopsis. We suggested that SFAR4 plays an important role in fatty acid degradation, thus reducing the fatty acid content.

Orchids (Oncidium and Phalaenopsis).

This chapter describes an efficient and reproducible method for large-scale propagation of Oncidium and Phalaenopsis protocorm-like bodies (PLBs) using floral stalk sections and seeds, respectively. The propagated PLBs can be used for Agrobacterium-mediated transformation. An advanced transformation system for Oncidium and Phalaenopsis orchids has been established. This protocol demonstrates that the time during which the PLBs are cocultivated with Agrobacterium is the key to promoting transformation efficiency. Modified DNA and RNA extraction methods are also provided to diminish polysaccharide contamination and to improve the quality for further molecular analysis.

Virus resistance in orchids.

Orchid plants, Phalaenopsis and Dendrobium in particular, are commercially valuable ornamental plants sold worldwide. Unfortunately, orchid plants are highly susceptible to viral infection by Cymbidium mosaic virus (CymMV) and Odotoglossum ringspot virus (ORSV), posing a major threat and serious economic loss to the orchid industry worldwide. A major challenge is to generate an effective method to overcome plant viral infection. With the development of optimized orchid transformation biotechnological techniques and the establishment of concepts of pathogen-derived resistance (PDR), the generation of plants resistant to viral infection has been achieved. The PDR concept involves introducing genes that is(are) derived from the virus into the host plant to induce RNA- or protein-mediated resistance. We here review the fundamental mechanism of the PDR concept, and illustrate its application in protecting against viral infection of orchid plants.

MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress.

Methionine sulfoxide reductases (MSRs) catalyse the reduction of oxidized methionine residues, thereby protecting proteins against oxidative stress. Accordingly, MSRs have been associated with stress responses, disease, and senescence in a taxonomically diverse array of organisms. However, the cytosolic substrates of MSRs in plants remain largely unknown. Here, we used a proteomic analysis strategy to identify MSRB7 substrates. We showed that two glutathione transferases (GSTs), GSTF2 and GSTF3, had fewer oxidized methionine (MetO) residues in MSRB7-overexpressing Arabidopsis thaliana plants than in wild-type plants. Conversely, GSTF2 and GSTF3 were highly oxidized and unstable in MSRB7-knockdown plants. MSRB7 was able to restore the MetO-GSTF2M100/104 and MetO-GSTF3M100 residues produced during oxidative stress. Furthermore, both GSTs were specifically induced by the oxidative stress inducer, methyl viologen. Our results indicate that specific GSTs are substrates of MSRs, which together provide a major line of defence against oxidative stress in A. thaliana.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families.

The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5'RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.