R2R3 MYB Research Articles

Transcriptome Analysis Reveals Candidate Genes Involved in Gibberellin-Induced Fruit Development in Rosa roxburghii.

Gibberellins (GAs) play indispensable roles in the fruit development of horticultural plants. Unfortunately, the molecular basis behind GAs regulating fruit development in R. roxburghii remains obscure. Here, GA3 spraying to R. roxburghii 'Guinong 5' at full-bloom promoted fruit size and weight, prickle development, seed abortion, ascorbic acid accumulation, and reduction in total soluble sugar. RNA-Seq analysis was conducted to generate 45.75 Gb clean reads from GA3- and non-treated fruits at 120 days after pollination. We obtained 4275 unigenes belonging to differently expressed genes (DEGs). Gene ontology and the Kyoto Encyclopedia of Genes and Genomes displayed that carbon metabolism and oxidative phosphorylation were highly enriched. The increased critical genes of DEGs related to pentose phosphate, glycolysis/gluconeogenesis, and citrate cycle pathways might be essential for soluble sugar degradation. Analysis of DEGs implicated in ascorbate revealed the myoinositol pathway required to accumulate ascorbic acid. Finally, DEGs involved in endogenous phytohormones and transcription factors, including R2R3 MYB, bHLH, and WRKY, were determined. These findings indicated that GA3-trigged morphological alterations might be related to the primary metabolites, hormone signaling, and transcription factors, providing potential candidate genes that could be guided to enhance the fruit development of R. roxburghii in practical approaches.

Vitis Myb14 confer cold and drought tolerance by activating lipid transfer protein genes expression and reactive oxygen species scavenge

The R2R3 Myb transcription factor exhibits a wide range of functions and participates in various biological processes in plant development, secondary metabolism, and abiotic stress tolerance, among others. Vitis Myb14 initially identified for its involvement in resveratrol synthesis in grapevines. In this study, we investigate its role in abiotic stress tolerance. Significant differences in expression were observed between two grape varieties, Vitis amurensis (Cold-hardy) and V. vinifera (Cold-sentitive), under abiotic and hormone treatments. Both VvMyb14 and VaMyb14 demonstrated responsiveness to cold, drought and high salt treatment, but VaMyb14 exhibited a quicker and more pronounced response. To investigate further, we overexpressed VaMyb14 in A. thalina and found that VaMyb14 OE plants showed significantly enhanced cold and drought tolerance compared to wild-type plants. Additionally, the transgenic lines exhibited increased antioxidant enzyme activity, particularly POD activity, and reduced MDA content. Microarray analysis of VaMyb14 OE plants revealed up-regulation of several ABA metabolism and signal transduction genes, including several LTPs, PP2Cs, RD29B, COR78 and other structural genes, indicating that VaMyb14 has the capacity to reprogram a significant signaling pathway. Furthermore, comparative mRNA sequencing profiling of 35S:VaMyb14 grapevine callus indicated its involvement its function involved in ROS scavenging and ABA signaling. These findings collectively demonstrate that Vitis Myb14 serves as a critical regulator in grapevine stress responses, contributing to improved defense against necrotrophic pathogens, enhanced phytoalexin resveratrol production, and increased drought or cold tolerance.

STRUCTURAL AND REGULATORY ASPECTS OF MORPHOGENESIS IN <i>EQUISETUM</i> <i>SYLVATICUM</i> AND <i>EQUISETUM</i> <i>FLUVIATILE</i> AND THE ISSUE OF HOMOLOGY OF LEAVES OF HORSETAILS AND OTHER FERNS

The structure of the shoot apical meristem (SAM), ultrastructure of its cells and the origin of primordia of organs in the SAM of horsetails Equisetum sylvaticum L. and E. fluviatile L. were studied. A relatively low degree of vacuolation and structural homogeneity of the cells that compose the surface initials zone (SI), the presence of single starch grains in their plastids and single lipid droplets in the cytoplasm of the SI cells were revealed. These features are more similar to those in the monoplex SAM of lycophytes than to those in leptosporangiate ferns. It is shown that initiation of leaves in E. sylvaticum and E. fluviatile via emergence of the leaf apical initials (LAIs) in the surface initials zone is similar to that of other plants with monoplex SAM; both ferns and lycophytes. The main peculiarity of organogenesis in horsetails is the origin of the leaf whorl as a single structure; each whorl develops from the triplet of the shoot apical initial merophytes. The inner cells of the emerging leaf whorl that belong to the single triplet of merophytes differentiate into the intercalary meristem. Each phytomer of E. sylvaticum and E. fluviatile is composed of the whorl of congenitally fused leaves and a whorl of buds that emerge in the axils of leaf bases located between the blades and thus alternate with leaves. Each bud bears a single root primordium; bud primordia develop on aerial shoots while root primordia – on the underground shoots. Termination of the leaf apical meristem functioning, that consequences from the vacuolation of the LAI, leads to the absence of the marginal meristem and the vascular tissues in the leaf blade. Proximodistal development of the leaf whorl and origin of buds results from meristematic activity of the peripheral part of the intercalary meristem. The search for the homologues of genes encoding known for angiosperms regulators of the development of adaxial (C3HDZ and ARP) and abaxial (YABBY and KANADI) leaf domains in available transcriptomes of horsetails and matching of the found homologues with that from the genomes of mosses, ferns and gymnosperms was undertaken in the context of assessment of presumptive regulatory similarities or differences of leaves of horsetails and other ferns in the context of leaf homology in the two groups. MEME analysis has shown that R2-R3 MYB proteins from transciptomes of horsetails are not ARP homologues. Thus it has been revealed that horsetail have single adaxial domain regulator (C3HDZ) and single abaxial domain regulator (KANADI), as other ferns. This finding suggests the probable loss of other regulators of the adaxial (ARP) and abaxial (YABBY) domains in the common ancestor of Polypodiophyta. Phylogenetic analysis of the identified homologues of genes that encode WOX proteins suggest that the T3 clade, that includes regulators of marginal (WOX3) and plate (WOX1) leaf meristems, originated in a common ancestor of Polypodiophyta, also indicating similarities in the molecular genetic regulation of the leaves in horsetails and other ferns.

Barley TAPETAL DEVELOPMENT and FUNCTION1 (HvTDF1) gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants.

The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley. Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics. Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots. This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration.

Dissected Leaf 1 encodes an MYB transcription factor that controls leaf morphology in potato.

The transcription factor StDL1 regulates dissected leaf formation in potato and the genotype frequency of recessive Stdl1/Stdl1, which results in non-dissected leaves, has increased in cultivated potatoes. Leaf morphology is a key trait of plants, influencing plant architecture, photosynthetic efficiency and yield. Potato (Solanum tuberosum L.), the third most important food crop worldwide, has a diverse leaf morphology. However, despite the recent identification of several genes regulating leaf formation in other plants, few genes involved in potato leaf development have been reported. In this study, we identified an R2R3 MYB transcription factor, Dissected Leaf 1 (StDL1), regulating dissected leaf formation in potato. A naturally occurring allele of this gene, Stdl1, confers non-dissected leaves in young seedlings. Knockout of StDL1 in a diploid potato changes the leaf morphology from dissected to non-dissected. Experiments in N. benthamiana and yeast show that StDL1 is a transcriptional activator. Notably, by calculating the genotype frequency of the Stdl1/Stdl1 in 373-potato accessions, we found that it increases significantly in cultivated potatoes. This work reveals the genetic basis of dissected leaf formation in potato and provides insights into plant leaf morphology.

CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum. However, the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear, which greatly restricts design of molecular breeding in chrysanthemum. Here, a chrysanthemum SG7 R2R3 MYB transcription factor (TF), CmMYB3-like, was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage. Quantitative real time PCR (qRT-PCR) assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage. Genetic transformation of chrysanthemum and dual-luciferase assays in N. benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly. However, overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols. Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation, but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage. Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

Functional characterization of DcMYB11, an R2R3 MYB associated with the purple pigmentation of carrot petiole.

DcMYB11, an R2R3 MYB gene associated with petiole anthocyanin pigmentation in carrot, was functionally characterized. A putative enhancer sequence is able to increase DcMYB11 activity. The accumulation of anthocyanin pigments can exhibit different patterns across plant tissues and crop varieties. This variability allowed the investigation of the molecular mechanisms behind the biosynthesis of these pigments in several plant species. Among crops, carrots have a well-defined anthocyanin pigmentation pattern depending on the genic background. In this work, we report on the discovery of DNA structural differences affecting the activity of an R2R3 MYB (encoded by DcMYB11) involved in anthocyanin regulation in carrot petiole. To this end, we first verified the function of DcMYB11 using heterologous systems and identified three different alleles which may explain differences in petiole pigmentation. Characterization of the DcMYB11 alleles at the 5' upstream sequence unveiled a sequence that functions as a putative enhancer. In conclusion, this study provides novel insight into the molecular mechanisms controlling anthocyanin accumulation in carrot. By these outcomes, we expanded our knowledge on the cis-regulatory sequences in plants.

Balancing selection on an MYB transcription factor maintains the twig trichome color variation in Melastoma normale

BackgroundThe factors that maintain phenotypic and genetic variation within a population have received long-term attention in evolutionary biology. Here the genetic basis and evolution of the geographically widespread variation in twig trichome color (from red to white) in a shrub Melastoma normale was investigated using Pool-seq and evolutionary analyses.ResultsThe results show that the twig trichome coloration is under selection in different light environments and that a 6-kb region containing an R2R3 MYB transcription factor gene is the major region of divergence between the extreme red and white morphs. This gene has two highly divergent groups of alleles, one of which likely originated from introgression from another species in this genus and has risen to high frequency (> 0.6) within each of the three populations under investigation. In contrast, polymorphisms in other regions of the genome show no sign of differentiation between the two morphs, suggesting that genomic patterns of diversity have been shaped by homogenizing gene flow. Population genetics analysis reveals signals of balancing selection acting on this gene, and it is suggested that spatially varying selection is the most likely mechanism of balancing selection in this case.ConclusionsThis study demonstrate that polymorphisms on a single transcription factor gene largely confer the twig trichome color variation in M. normale, while also explaining how adaptive divergence can occur and be maintained in the face of gene flow.

Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium ‘Siberia’

Terpenoids are the main components contributing to the fragrance of Lilium ‘Siberia’, and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids. Although the major terpene synthases in Lilium ‘Siberia’ have been identified, how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower. This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium, and disclose the related underlying transcriptional regulation mechanism. In this study, we identified three R2R3-MYB TFs—LiMYB1, LiMYB305 and LiMYB330, which were involved in regulating the biosynthesis of terpenes in Lilium ‘Siberia’. Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds. When LiMYB1, LiMYB305 and LiMYB330 were overexpressed in flowers, the release of some main monoterpenes, such as linalool and ocimene, as well as the expression of TPS genes, especially for LiTPS2, were enhanced. A virus-induced gene silencing (VIGS) assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes. The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2. Moreover, the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330, indicating their synergistic roles in the regulation of floral terpene biosynthesis. In general, these results indicated that LiMYB1, LiMYB305, and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.

Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

PpMYB44 positively affects salicylic acid biosynthesis in Pichia guilliermondii-induced peach fruit resistance against Rhizopus stolonifer

Antagonistic yeast induces postharvest disease resistance and delays fruit decay. Salicylic acid (SA), a signal molecule, is required for activating induced disease resistance in various harvested fruit. However, there is limited information about the transcriptional regulatory mechanism of SA biosynthesis in response to Rhizopus stolonifer in peaches. Our results showed that Pichia guilliermondii application promoted SA accumulation accompanied by increasing SA biosynthetic enzyme activity of PAL and ICS and upregulating transcripts of SA synthesis-related genes and defense genes, including PpPAL1, PpPAL2, PpICS1, PpICS2, PpCM1, PpCM2, PpCM3, PpBA2H, PpSARD1, PpCBP60g, PpPR1, PpPR2, and PpPR5. In addition, an R2R3 MYB transcription factor, PpMYB44, was induced in P. guilliermondii-peach fruit in response to fungal disease, and PpMYB44 was identified to mediate SA synthesis by binding to the promoters of PpPAL1 and PpSARD1. Furthermore, the transient overexpression of PpMYB44 in peach fruit reduced disease incidence and severity, upregulated the expression of SA synthesis-related genes (PpPAL1 and PpSARD1) and enhanced SA accumulation. Silencing of PpMYB44 in peach fruit increased disease incidence and severity, downregulated the transcript levels of PpPAL1 and PpSARD1, and decreased SA accumulation. Thus, the study indicates that PpMYB44 regulates yeast-induced disease resistance by increasing the SA content in peaches.

BcMYB111 Responds to BcCBF2 and Induces Flavonol Biosynthesis to Enhance Tolerance under Cold Stress in Non-Heading Chinese Cabbage.

Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3-MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H2O2 content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2-BcMYB111-BcF3H/BcFLS1 in NHCC.

NataMYB4, a flower specific gene, regulates the flavonoid biosynthesis in Chinese Narcissus

R2R3 MYBs play a crucial role in flower development and its associated flavonoid biosynthesis, but their regulatory network in Chinese Narcissus is not fully understood. Anthocyanin, flavonol and proanthocyanin are very important secondary metabolites with various biological functions and their biosynthesis is controlled by MYB transcriptional factors. NataMYB4, as a member of the R2R3-MYB gene family, isolated from Chinese Narcissus, was found mainly expressed in flowers, particularly in petals and corona. In addition to the R2-R3 region, NataMYB4 displays a conserved motif “NF/YWSV/MEDF/LW” at the end of the C-terminal. Transient expression in tobacco leaves showed that NataMYB4 significantly decreased the red pigmentation that was induced by an anthocyanin activator StMYB. Ectopic overexpression of NataMYB4 in tobacco altered the flower color by reducing the pigmentation and anthocyanin contents in transgenic flowers. However, the contents of total phenolic acid and total flavonol were increased in the transgenic tobacco lines. qPCR analysis indicated that transcript levels of anthocyanin biosynthesis genes were down-regulated in transgenic flowers carrying the NataMYB4 gene, while the flavonol biosynthetic pathway key gene FLS showed higher expression level in transgenic tobacco flowers. Dual luciferase assays showed that co-infiltration of NataMYB4 and NatabHLH1 significantly activated the promoter of the Chinese Narcissus FLS gene. Furthermore, a yeast two-hybrid assay confirmed that NataMYB4 interacts with NatabHLH1. The outcomes of our effort in this study provide novel insights into the regulation of flavonoid biosynthetic pathways in Chinese Narcissus.

An R2R3 MYB transcription factor PsFLP regulates the symmetric division of guard mother cells during stomatal development in Pisum sativum.

MYB transcriptional regulators belong to one of the most significant transcription factors families in plants, among which R2R3-MYB transcription factors are involved in plant growth and development, hormone signal transduction, and stress response. Two R2R3-MYB transcription factors, FLP and its paralogous AtMYB88, redundantly regulate the symmetrical division of guard mother cells (GMCs), and abiotic stress response in Arabidopsis thaliana. Only one orthologue gene of FLP was identified in pea (Pisum sativum FLP; PsFLP). In this study, we explored the gene function of PsFLP by virus-induced gene silencing (VIGS) technology. The phenotypic analysis displayed that the silencing of PsFLP expression led to the abnormal development of stomata and the emergence of multiple guard cells tightly united. In addition, the abnormal stomata of flp could be fully rescued by PsFLP driven by the FLP promoter. In conclusion, the results showed that PsFLP plays a conservative negative role in regulating the symmetric division of GMC during stomatal development. Based on real-time quantitative PCR, the relative expressions of AAO3, NCED3, and SnRK2.3 significantly increased in the flp pFLP::PsFLP plants compared to mutant, indicating that PsFLP might be involved in drought stress response. Thus, PsFLP regulates the genes related to cell cycle division during the stomatal development of peas and participates in response to drought stress. The study provides a basis for further research on its function and application in leguminous crop breeding.

Mechanistic and structural insight into R2R3-MYB transcription factor in plants: molecular dynamics based binding free energy analysis

The MYB transcription factor (TF) family is essential for various plant growth and development processes, including responses to biotic and abiotic stresses. This study investigated the R2R3-MYB protein structure from five plants, including cereal crops. The R2R3–MYB protein structure was docked with the DNA structure, and the best complexes were selected for two runs of molecular dynamics (MD) simulations to investigate the key interacting residues and conformational changes in the R2R3-MYB proteins caused by DNA binding. The MM/PBSA method calculated the binding free energy for each R2R3–MYB protein–DNA complex, showing strong interaction. Hydrophobic and hydrogen bonds significantly stabilized the R2R3–MYB protein–DNA complexes. The principal component analysis showed high restrictions on the movement of protein atoms in the phase space. A similar MD simulation analysis was performed using the crystal structure of the R2R3–MYB protein–DNA complex from Arabidopsis thaliana, and the generated complexes resembled the X-ray crystal structure. This is the first detailed study on the R2R3–MYB protein–DNA complex in cereal crops, providing a cost-effective solution to identify the key interacting residues and analyze the conformational changes in the MYB domain before and after DNA binding. Communicated by Ramaswamy H. Sarma

An R2R3 MYB gene GhMYB3 functions in drought stress by negatively regulating stomata movement and ROS accumulation

MYB transcription factors are one of the largest TF families involved in plant growth and development as well as biotic and abiotic stresses. In this study, we report the identification and functional characterization of a stress-responsive MYB gene (GhMYB3) from drought stress related transcriptome of upland cotton. GhMYB3, belonging to the R2R3-type, has high sequence similarity with AtMYB3 and was localized in the nucleus. Silence of GhMYB3 enhanced the drought tolerance of cotton seedlings and plants, reduced the water loss rate, and enhanced stomatal closure. In addition, GhMYB3i lines exhibited less ROS accumulation, as well as higher antioxidant enzyme activity and increased content of anthocyanins and proanthocyanidins than WT plants after drought stress. The expression level of flavonoid biosynthesis- and stress-related genes were up-regulated in GhMYB3i lines under drought stress condition. These results demonstrated that GhMYB3 acted as a negative regulator in upland cotton response to drought stress by regulating stomatal closure and ROS accumulation.

Novel R2R3 MYB transcription factors regulate anthocyanin synthesis in Aubergine tomato plants

BackgroundA high content in anthocyanins, for their health beneficial properties, represents an added value for fruits and vegetables. Tomato (Solanum lycopersicum) is one of the most consumed vegetables worldwide and is rich in vitamins and carotenoids. In recent years, purple-skinned tomatoes, enriched of anthocyanins, were produced recovering allelic variants from wild Solanum species. The molecular basis of the Anthocyanin fruit (Aft) locus, exploited by breeders to activate the anthocyanin synthesis in tomato epicarp, has been recently identified in the correct splicing of the R2R3 MYB gene AN2like. Aubergine (Abg) is a tomato accession which introgressed from Solanum lycopersicoides a locus activating the synthesis of anthocyanins in the fruit. The Abg locus was mapped in the region of chromosome 10 containing Aft and the possibility that Abg and Aft represented alleles of the same gene was hypothesized.ResultsWe dissected the R2R3 MYB gene cluster located in the Abg genomic introgression and demonstrated that AN2like is correctly spliced in Abg plants and is expressed in the fruit epicarp. Moreover, its silencing specifically inhibits the anthocyanin synthesis. The Abg allele of AN2like undergoes alternative splicing and produces two proteins with different activities. Furthermore, in Abg the master regulator of the anthocyanin synthesis in tomato vegetative tissues, AN2, is very poorly expressed. Finally, a novel R2R3 MYB gene was identified: it encodes another positive regulator of the pathway, whose activity was lost in tomato and in its closest relatives.ConclusionIn this study, we propose that AN2like is responsible of the anthocyanin production in Abg fruits. Unlike wild type tomato, the Abg allele of AN2like is active and able to regulate its targets. Furthermore, in Abg alternative splicing leads to two forms of AN2like with different activities, likely representing a novel type of regulation of anthocyanin synthesis in tomato.

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

OsCCRL1 is Essential for Phenylpropanoid Metabolism in Rice Anthers

Phenylpropanoid metabolism and timely tapetal degradation are essential for anther and pollen development, but the underlying mechanisms are unclear. In the current study, to investigate this, we identified and analyzed the male-sterile mutant, osccrl1 (cinnamoyl coA reductase-like 1), which exhibited delayed tapetal programmed cell death (PCD) and defective mature pollen. Map-based cloning, genetic complementation, and gene knockout revealed that OsCCRL1 corresponds to the gene LOC_Os09g32020.2, a member of SDR (short-chain dehydrogenase/reductase) family enzyme. OsCCRL1 was preferentially expressed in the tapetal cells and microspores, and localized to the nucleus and cytoplasm in both rice protoplasts and Nicotiana benthamiana leaves. The osccrl1 mutant exhibited reduced CCRs enzyme activity, less lignin accumulation, delayed tapetum degradation, and disrupted phenylpropanoid metabolism. Furthermore, an R2R3 MYB transcription factor OsMYB103/OsMYB80/OsMS188/BM1, involved in tapetum and pollen development, regulates the expression of OsCCRL1. Finally, the osmyb103 osccrl1 double mutants, exhibited the same phenotype as the osmyb103 single mutant, further indicating that OsMYB103/OsMYB80/OsMS188/BM1 functions upstream of OsCCRL1. These findings help to clarify the role of phenylpropanoid metabolism in male sterility and the regulatory network underlying the tapetum degradation.

Reduced glucosinolate content in oilseed rape (Brassica napus L.) by random mutagenesis of BnMYB28 and BnCYP79F1 genes

The presence of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal severely restricts its utilization as animal feed. Therefore, reducing the GSL content to < 18 µmol/g dry weight in the seeds is a major breeding target. While candidate genes involved in the biosynthesis of GSLs have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 encoding an R2R3 MYB transcription factor and a cytochrome P450 enzyme, respectively, we aimed to reduce the seed GSL content in rapeseed. After expression analyses on single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed ‘Express617’ to detect functional mutations in the two gene families. Our results provide the first functional analysis by knock-out for the two GSL biosynthesis genes in winter rapeseed. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced seed aliphatic GSLs, primarily progoitrin. Compared to the wildtype Express617 control plants (36.3 µmol/g DW), progoitrin levels were decreased by 55.3% and 32.4% in functional mutants of BnMYB28 (16.20 µmol/g DW) and BnCYP79F1 (24.5 µmol/g DW), respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future.