Moniliophthora perniciosa is the causal agent of the witches' broom disease of cacao (Theobroma cacao), and it can infect the tomato (Solanum lycopersicum) 'Micro-Tom' (MT) cultivar. Typical symptoms of infection are stem swelling and axillary shoot outgrowth, whereas reduction in root biomass is another side effect. Using infected MT, we investigated whether impaired root growth derives from hormonal imbalance or sink competition. Intense stem swelling coincided with a reduction in root biomass, predominantly affecting lateral roots. RNA-seq analyses of root samples identified only a few differentially expressed genes involved in hormone metabolism, and root hormone levels were not expressively altered. Inoculation of the auxin highly-sensitive entire mutant genotype maintained the impaired root phenotype; in contrast, the low-cytokinin MT transgenic line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2) with fewer symptoms did not exhibit root growth impairment. Genes involved in cell wall, carbohydrate, and amino acid metabolism were downregulated, accompanied by lower levels of carbohydrate and amino acid in roots, suggesting a reduction in metabolite availability. 13CO2 was supplied to MT plants, and less 13C was detected in the roots of infected MT but not in those of 35S::AtCKX2 line plants, suggesting that cytokinin-mediated sugar sink establishment at the infection site may contribute to impaired root growth. Exogenous sucrose application to roots of infected MT plants partially restored root growth. We propose that the impairment of lateral root development is likely attributed to disrupted sugar signalling and photoassimilate supply by establishing a strong sugar sink at the infected stem.

Fruit morphogenesis is determined by the coordination of cell division and expansion, which are fundamental processes required for the development of all plant organs. Here, we show that the regulation of TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) LANCEOLATE (TCP2/LA) by miR319 is crucial for tomato fruit morphology. The loss of miR319 regulation in the semi-dominant La mutant led to a premature SlTCP2/LA expression during gynoecium patterning, which results in modified cell division during carpel development. As a consequence, La mutants exhibited elongated ovary and fruit shape, and a reduced number of ovules and seeds. Elongated fruit shape in La may be partially due to the SlTCP2/LA-mediated repression of OVATE activity in young floral buds. Further analysis showed that the de-repression of SlTCP2/LA decreases auxin responses in young floral buds by directly repressing SlYUCCA4 expression, but SlTCP2/LA also acts in parallel with ENTIRE (E) to orchestrate fruit morphology and seed production. Our study defines a novel miRNA-based molecular link between the domestication-associated OVATE gene and auxin responses. Given the striking variation in fruit morphology among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of auxin dynamics may be a common driver in the evolution of fruit shape diversity.

Article title: Tomato miR156-targeted SlSBP15 represses shoot branching via modulating hormone dynamics and interacting with GOBLET and BRANCHED1b Authors: Carlos Hernán Barrera-Rojas, Mateus Henrique Vicente, Diego Armando Pinheiro Brito, Eder M. Silva, Aitor Munoz Lopez, Leticia F. Ferigolo, Rafael Monteiro do Carmo, Carolina M. S. Silva, Geraldo F.F. Silva, Joao P. O. Correa, Marcela M. Notini, Luciano Freschi, Pilar Cubas, Fabio T.S. Nogueira

ABSTRACT Many developmental processes associated with fruit development take place 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 that GA and miR156 function in tomato FM and fruit patterning. High GA responses or overexpression of miR156 (156OE), which leads to low levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN– LIKE ( SPL/SBP ), resulted in enlarged FMs, defects in FM determinacy and fruits with increased locule number. Conversely, low GA responses reduced fruit indeterminacy and locule number, and overexpression of a miR156-resistant SlSBP15 allele ( rSBP15 ) reduced cell number and size in the FM, as well as locule number. GA responses were partially required for the fruit defects observed in 156OE and rSBP15 plants. 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 that the miR156/ SlSBP /GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to genetically fine-tune aspects of fruit development that have been important for tomato domestication.

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.

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.

Diversity in fruit morphology is one of the hallmarks of varietal differences among modern cultivars of fruit-bearing crops. As evolutionarily related organs, fruits and leaves share developmental processes, but there are surprisingly few connections between regulatory pathways for fruit and leaf development. Here, we show the regulation of the leaf development-associated TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP4/LANCEOLATE (TCP4/LA) transcription factor by the microRNA319 (miR319) is crucial for gynoecium patterning and establishment of fruit morphology. Loss of miR319 regulation leads to a premature, ectopic TCP4/LA expression during gynoecium patterning, which results in elongated fruits, resembling ovate mutants. TCP4/LA modulates tomato fruit development and morphology partially by directly repressing OVATE expression as early as 5-8 days post-inflorescence (dpi) flower buds. Furthermore, miR319-targeted CINCINNATA-like TCP4/LANCEOLATE controls auxin responses in developing flower buds by directly binding to the SlYUCCA4 promoter. Modulation of auxin biosynthesis by TCP4/LA is shared with other CINCINNATA-like TCPs during Arabidopsis gynoecium patterning. Our study defines a novel miRNA-based molecular link between OVATE , a fundamental gene associated with tomato domestication, and auxin responses in the control of fruit development and morphology. Given the striking variation in fruit shape among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of hormone dynamics may be a common driver in the evolution of fruit-shape diversity. ### Competing Interest Statement The authors have declared no competing interest.

SUMMARYDiversity in fruit morphology is one of the hallmarks of varietal differences among modern cultivars of fruit-bearing crops. As evolutionarily related organs, fruits and leaves share developmental processes, but there are surprisingly few connections between regulatory pathways for fruit and leaf development. Here, we show the regulation of the leaf development-associated TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP4/LANCEOLATE (TCP4/LA) transcription factor by the microRNA319 (miR319) is crucial for gynoecium patterning and establishment of fruit morphology. Loss of miR319 regulation leads to a premature, ectopic TCP4/LA expression during gynoecium patterning, which results in elongated fruits, resembling ovate mutants. TCP4/LA modulates tomato fruit development and morphology partially by directly repressing OVATE expression as early as 5-8 days post-inflorescence (dpi) flower buds. Furthermore, miR319-targeted CINCINNATA-like TCP4/LANCEOLATE controls auxin responses in developing flower buds by directly binding to the SlYUCCA4 promoter. Modulation of auxin biosynthesis by TCP4/LA is shared with other CINCINNATA-like TCPs during Arabidopsis gynoecium patterning. Our study defines a novel miRNA-based molecular link between OVATE, a fundamental gene associated with tomato domestication, and auxin responses in the control of fruit development and morphology. Given the striking variation in fruit shape among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of hormone dynamics may be a common driver in the evolution of fruit-shape diversity.

Diversity in fruit morphology is one of the hallmarks of varietal differences among modern cultivars of fruit-bearing crops. As evolutionarily related organs, fruits and leaves share developmental processes, but there are surprisingly few connections between regulatory pathways for fruit and leaf development. Here, we show the regulation of the leaf development-associated TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP4/LANCEOLATE (TCP4/LA) transcription factor by the microRNA319 (miR319) is crucial for gynoecium patterning and establishment of fruit morphology. Loss of miR319 regulation leads to a premature, ectopic TCP4/LA expression during gynoecium patterning, which results in elongated fruits, resembling ovate mutants. TCP4/LA modulates tomato fruit development and morphology partially by directly repressing OVATE expression as early as 5-8 days post-inflorescence (dpi) flower buds. Furthermore, miR319-targeted CINCINNATA-like TCP4/LANCEOLATE controls auxin responses in developing flower buds by directly binding to the SlYUCCA4 promoter. Modulation of auxin biosynthesis by TCP4/LA is shared with other CINCINNATA-like TCPs during Arabidopsis gynoecium patterning. Our study defines a novel miRNA-based molecular link between OVATE, a fundamental gene associated with tomato domestication, and auxin responses in the control of fruit development and morphology. Given the striking variation in fruit shape among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of hormone dynamics may be a common driver in the evolution of fruit-shape diversity.

The microRNA156 (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 hub modulates tomato shoot branching (SB) by connecting phytohormones with important genetic pathways regulating both axillary bud (AB) development and outgrowth. We verified that plants overexpressing the miR156 (156-OE plants) display high SB, whereas plants overexpressing a miR156-resistant SlSBP15 alelle (rSBP15 plants) display arrested SB and are able to partially restore the wild-type (WT) phenotype in156-OE background. Although rSBP15 plants showed ABs smaller than MT, its activation is dependent on shoot apex-derived auxin transport inhibition. Additionally, hormonal measurements reveal that IAA and ABA concentrations were lower in 156-OE and higher in rSBP15-OE plants. SlSBP15 regulates AB development and outgrowth by inhibiting auxin transport and the activity of GOBLET ( GOB ), and by interacting with BRANCHED1b (SlBRC1b) at the protein level to control abscisic acid (ABA) levels within ABs. Our data provide a new mechanism by which the miR156/ SPL/SBP hub regulates SB, and suggest that SlSBP15 has potential applications in improving tomato architecture.

Witches’ broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Fruits are defined as mature ovaries originated from a coordinated development between ovules (precursors of seeds) and carpels (precursors of the fruit body). Disconnecting the ovule development from carpel development is a long-desired goal in agriculture to generate seedless fruits easier to consume. However, pollination and fertilization are required to promote hormone pathways—i.e., auxin and gibberellin—that are crucial for seed and fruit set (Gillaspy et al., 1993; Raghavan, 2003). Even though developing seeds are central for normal fruit development, examples of seedless or parthenocarpic fruits are present in wild and cultivated species (Joldersma and Liu, 2017).

Fruits are originated from the transition of a quiescent ovary to a fast-growing young fruit. The evolution of reproductive structures such as ovary and fruit has made seed dispersal easier, which is a key process for reproductive success in flowering plants. The complete fruit development and ripening are characterized by a remarkable phenotypic plasticity which is orchestrated by a myriad of genetic factors. In this context, transcriptional regulation by non-coding small (i.e., microRNAs) and long (lncRNAs) RNAs underlies important mechanisms controlling reproductive organ development. These mechanisms may act together and interact with other pathways (i.e., phytohormones) to regulate cell fate and coordinate reproductive organ development. Functional genomics has shown that non-coding RNAs regulate a diversity of developmental reproductive stages, from carpel formation and ovary development to the softening of the ripe/ripened fruit. This layer of transcriptional control has been associated with ovule, seed, and fruit development as well as fruit ripening, which are crucial developmental processes in breeding programs because of their relevance for crop production. The final ripe fruit is the result of a process under multiple levels of regulation, including mechanisms orchestrated by microRNAs and lncRNAs. Most of the studies we discuss involve work on tomato and Arabidopsis. In this review, we summarize non-coding RNA-controlled mechanisms described in the current literature that act coordinating the main steps of gynoecium development/patterning and fruit ripening.

Age-regulated microRNA156 (miR156) and targets similarly control the competence to flower in diverse species. By contrast, the diterpene hormone gibberellin (GA) and the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote flowering in the facultative long-day Arabidopsis thaliana, but suppress it in the day-neutral tomato (Solanum lycopersicum). We combined genetic and molecular studies and described a new interplay between GA and two unrelated miRNA-associated pathways that modulates tomato transition to flowering. Tomato PROCERA/DELLA activity is required to promote flowering along with the miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL/SBP) transcription factors by activating SINGLE FLOWER TRUSS (SFT) in the leaves and the MADS-Box gene APETALA1(AP1)/MC at the shoot apex. Conversely, miR319-targeted LANCEOLATE represses floral transition by increasing GA concentrations and inactivating SFT in the leaves and AP1/MC at the shoot apex. Importantly, the combination of high GA concentrations/responses with the loss of SPL/SPB function impaired canonical meristem maturation and flower initiation in tomato. Our results reveal a cooperative regulation of tomato floral induction and flower development, integrating age cues (miR156 module) with GA responses and miR319-controlled pathways. Importantly, this study contributes to elucidate the mechanisms underlying the effects of GA in controlling flowering time in a day-neutral species.

BACKGROUND Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane-bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. RESULTS We present the first study aimed at developing novel fungal AOX inhibitors. N-Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j-41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j-41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure-activity relationships analyses. CONCLUSION We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry.

The exceptional abilities of stink bugs (Hemiptera: Pentatomidae) to colonize a diverse group of plants have been attributed to the feeding behaviors and the functions of the salivary complex of these insects. Here, we describe the ultrastructure of the salivary glands of the Neotropical brown stink bug, Euschistus heros, which is a major component of the pentatomid pest complex on soybeans, Glycine max, in the neotropics. Our results revealed a salivary gland complex consisting of two lobes (i.e., anterior and posterior), with a constriction between them (i.e., the hilum), in which the salivary and accessory gland ducts are inserted. The principal gland epithelium has a single layer of cells lining an enlarged lumen filled with saliva, and these cells are cuboidal, rich in rough endoplasmic reticulum and secretory vesicles, with well-developed nuclei, all of which are typical features of protein-secreting cells. We report, for the first time in insects, the presence of a layer of muscle cells surrounding the columnar hilum epithelium. The accessory salivary gland cells are cuboidal with nuclei containing condensed chromatin and cytoplasm rich in vacuoles and rough endoplasmic reticulum, indicating the potential involvement of these glands in water transport/secretion. The lumen content of each lobe of the principal gland suggests that the lobes produce different compounds. Thus, our results suggest that the E. heros salivary complex might have unconventional mechanisms to mix/release saliva, which might help explain the polyphagous abilities of these insects.

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.

Senescence is the process that marks the end of a leaf's lifespan. As it progresses, the massive macromolecular catabolism dismantles the chloroplasts and, consequently, decreases the photosynthetic capacity of these organs. Thus, senescence manipulation is a strategy to improve plant yield by extending the leaf's photosynthetically active window of time. However, it remains to be addressed if this approach can improve fleshy fruit production and nutritional quality. One way to delay senescence initiation is by regulating key transcription factors (TFs) involved in triggering this process, such as the NAC TF ORESARA1 (ORE1). Here, three senescence-related NAC TFs from tomato (Solanum lycopersicum) were identified, namely SlORE1S02, SlORE1S03, and SlORE1S06. All three genes were shown to be responsive to senescence-inducing stimuli and posttranscriptionally regulated by the microRNA miR164 Moreover, the encoded proteins interacted physically with the chloroplast maintenance-related TF SlGLKs. This characterization led to the selection of a putative tomato ORE1 as target gene for RNA interference knockdown. Transgenic lines showed delayed senescence and enhanced carbon assimilation that, ultimately, increased the number of fruits and their total soluble solid content. Additionally, the fruit nutraceutical composition was enhanced. In conclusion, these data provide robust evidence that the manipulation of leaf senescence is an effective strategy for yield improvement in fleshy fruit-bearing species.

BACKGROUND:MicroRNAs (miRNAs) are important regulatory elements of gene expression. Similarly to coding genes, miRNA genes follow a birth and death pattern of evolution likely reflecting functional relevance and divergence. For instance, miRNA529 is evolutionarily related to miRNA156 (a highly conserved miRNA in land plants), but it is lost in Arabidopsis thaliana. Interestingly, both miRNAs target sequences overlap in some members of the SQUAMOSA promoter-binding protein like (SPL) family, raising important questions regarding the diversification of the miR156/miR529-associated regulatory network in land plants.RESULTS:In this study, through phylogenic reconstruction of miR156/529 target sequences from several taxonomic groups, we have found that specific eudicot SPLs, despite miRNA529 loss, retained the corresponding target site. Detailed molecular evolutionary analyses of miR156/miR529-target sequence showed that loss of miR529 in core eudicots, such as Arabidopsis, is correlated with a more relaxed selection of the miRNA529 specific target element, while miRNA156-specific target sequence is under stronger selection, indicating that these two target sites might be under distinct evolutionary constraints. Importantly, over-expression in Arabidopsis of MIR529 precursor from a monocot, but not from a basal eudicot, demonstrates specific miR529 regulation of AtSPL9 and AtSPL15 genes, which contain conserved responsive elements for both miR156 and miR529.CONCLUSIONS:Our results suggest loss of functionality of MIR529 genes in the evolutionary history of eudicots and show that the miR529-responsive element present in some eudicot SPLs is still functional. Our data support the notion that particular miRNA156 family members might have compensated for the loss of miR529 regulation in eudicot species, which concomitantly may have favored diversification of eudicot SPLs.