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Summary: Nicotianamine (NA) plays a crucial role in transporting metal ions, including iron (Fe), in plants; therefore, NICOTIANAMINE SYNTHASE (NAS) genes, which control NA synthesis, are tightly regulated at the transcriptional level. However, the transcriptional regulatory mechanisms of NAS genes require further investigations.In this study, we determined the role of bZIP44 in mediating plant response to Fe deficiency stress by conducting transformation experiments and assays.bZIP44 positively regulated the response of Arabidopsis to Fe deficiency stress by interacting with MYB10 and MYB72 to enhance their abilities to bind at NAS2 and NAS4 promoters, thereby increasing NAS2 and NAS4 transcriptional levels and promote NA synthesis.In summary, the transcription activities of bZIP44, MYB10, and MYB72 were induced in response to Fe deficiency stress, which enhanced the interaction between bZIP44 and MYB10 or MYB72 proteins, synergistically activated the transcriptional activity of NAS2 and NAS4, promoted NA synthesis, and improved Fe transport, thereby enhancing plant tolerance to Fe deficiency stress. [ABSTRACT FROM AUTHOR]

Chinese plum (Prunus salicina Lindl.) originates from China and makes a large contribution to the global production of plums. The P. salicina ‘Wushancuili’ has a green coloration and high fruit quality and is economically important in eliminating poverty and protecting ecology in the Yangtze River Three Gorges Reservoir. However, rain-induced cracking (rain-cracking, literally skin cracking caused by rain) is a limitation to ‘Wushancuili’ fruit production and causes severe losses. This study reported a high-quality ‘Wushancuili’ genome assembly consisting of a 302.17-Mb sequence with eight pseudo-chromosomes and a contig N50 of 23.59 Mb through the combination of Illumina sequencing, Pacific Biosciences HiFi Ⅲ sequencing, and high-throughput chromosome conformation capture technology. A total of 25 109 protein-coding genes are predicted and 54.17% of the genome is composed of repetitive sequences. ‘Wushancuili’ underwent a remarkable orthoselection during evolution. Gene identification revealed that loss-of-function in four core MYB10 genes results in the anthocyanin deficiency and absence of red color, revealing the green coloration due to the residual high chlorophyll in fruit skin. Besides, the occurrence of cracking is assumed to be closely associated with cell wall modification and frequently rain-induced pathogen enrichment through transcriptomic analysis. The loss of MYB10 genes might render fruit more susceptible to pathogen-mediated cracking by weakening the epidermal strength and reactive oxygen species (ROS) scavenging. Our findings provided fundamental knowledge regarding fruit coloration and rain-cracking and will facilitate genetic improvement and cultivation management in Chinese plums.

The R2R3-MYB transcription factor FveMYB10 is a major regulator of anthocyanin pigmentation in the red fruits of strawberry. fvemyb10 loss-of-function mutants form yellow fruits but still accumulate purple-colored anthocyanins in the petioles, suggesting that anthocyanin biosynthesis is under distinct regulation in fruits and petioles. From chemical mutagenesis in the diploid wild strawberry Fragaria vesca , we identified a green petioles (gp) -1 mutant that lacks anthocyanins in petioles. Using mapping-by-sequencing and transient functional assays, we confirmed that the causative mutation resides in a FveMYB10-Like (FveMYB10L) gene and that FveMYB10 and FveMYB10L function independently in the fruit and petiole, respectively. In addition to their tissue-specific regulation, FveMYB10 and FveMYB10L respond differently to changes in light quality, produce distinct anthocyanin compositions, and preferentially activate different downstream anthocyanin biosynthesis genes in their respective tissues. This work identifies a new regulator of anthocyanin synthesis and demonstrates that two paralogous MYB genes with specialized functions enable tissue-specific regulation of anthocyanin biosynthesis in fruit and petiole tissues. [ABSTRACT FROM AUTHOR]

Apple is a major fruit crop grown worldwide and provides humans with an essential diet and health benefits. One of the health benefits is related to the accumulation of fruit anthocyanin, which also provides fruit with an attractive red colour. It is known that an MdMYB10 allele containing a transposable element (TE) insertion in its promoter (termed Red-TE allele) underlies anthocyanin accumulation and red colouration in the fruit skin of cultivated apples. However, the distribution of this Red-TE allele in wider Malus germplasm accessions is not clear. In this study, we showed that MdMYB10 RNA in fruit skin was specifically expressed from the Red-TE allele by using allele-specific expression analysis of transcriptome data. Apple cultivars and hybrids with homozygous Red-TE alleles showed stronger red colour than those with heterozygous alleles after analysing 65 cultivars and 337 hybrids. Furthermore, both hetero- and homozygous plants growing in the same high-temperature conditions had different colourations. However, the Red-TE allele was not detected in 16 wild apple accessions showing red skin, indicating that the red skin colour of these wild apples was not conferred by the Red-TE allele. These findings provide guidance for selecting cultivars able to develop consistent red colouration under high growth temperature conditions and open the opportunity for identifying novel genetic variants underpinning fruit red colouration in wild apple species. [ABSTRACT FROM AUTHOR]

Low-nitrogen (LN) conditions typically promote anthocyanin biosynthesis in plants. Although previous studies primarily explored structural genes and transcription factors involved in anthocyanin biosynthesis, the role of long noncoding RNAs (lncRNAs) in this process under LN conditions remains less understood. This study identified LNC159c as a potential regulator of anthocyanin biosynthesis, with its expression notably downregulated under LN conditions. LNC159c functions as the host gene of miR159c, which regulates its expression. A dual-luciferase reporter assay and GFP signal detection demonstrated that miR159c inhibited MsMYB10 expression through base complementary pairing. Yeast one-hybrid (Y1H) and dual-LUC reporter assays jointly verified that MsMYB10 binds to the F3'H promoter. Stable overexpression in apple calli and transient overexpression in apple fruit peels and M. spectabilis leaves showed that LNC159c overexpression promoted miR159c expression, while inhibiting MsMYB10 expression and anthocyanin biosynthesis. MsMYB10 overexpression promoted anthocyanin biosynthesis, whereas the opposite results were observed in MsMYB10-silenced tissues. Thus, under LN conditions, reduced LNC159c expression limits miR159c production, relieving MsMYB10 inhibition and enhancing anthocyanin accumulation. These findings highlight the regulatory role of lncRNAs in anthocyanin biosynthesis under LN conditions., (© 2025 John Wiley & Sons Ltd.)

Background: Genome complexity is largely linked to diversification and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non-duplicated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identification and characterization of complex variability between genomes for plant breeding. Results: Here we improve and demonstrate the use of CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4.5% and a depth per sample of 11.9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region. Conclusions: CRISPR-Cas9 enrichment is a versatile and powerful tool for long-read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed. [ABSTRACT FROM AUTHOR]

Abstract Background Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. Results We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologies Cleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. Conclusions We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

SUMMARY: IRON‐REGULATED TRANSPORTER1 (IRT1) is the root high‐affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root‐to‐shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1‐2, compared to the wild type. Shoots of irt1‐2 are severely Fe‐deficient according to Fe content and marker transcripts, as expected. We generated irt1‐2 lines producing IRT1 mutant variants carrying single amino‐acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short‐term 55Fe uptake rates were uninformative concerning IRT1‐mediated transport. Overall irt1‐like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1‐mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis of irt1‐2, as well as root‐to‐shoot Fe partitioning and gene expression defects of irt1‐2, all of which are fully complemented by wild‐type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root‐to‐shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1‐dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops. [ABSTRACT FROM AUTHOR]

Secondary cell wall (SCW) formation is regulated by a multilevel transcriptional regulatory network, in which MYB transcription factors (TFs) play key roles. In woody plants, hundreds of MYB TFs have been identified, most of which have unknown functions in wood SCW biosynthesis. Here, we characterized the function of a Populus MYB gene, PtoMYB10. PtoMYB10 was found to encode an R2R3-MYB TF and exhibit dominant expression in xylem tissues. PtoMYB10 was determined to be located in the nucleus with the ability to activate transcription. Overexpression of PtoMYB10 in Populus resulted in a drastic increase in SCW thickening in xylem fiber cells as well as ectopic deposition of lignin in cortex cells. The expression of genes associated with lignin biosynthesis was induced in PtoMYB10 overexpressing plants, whereas repressed gene expression was found with the anthocyanin biosynthesis pathway. Lignin and anthocyanin are both produced from metabolites of the phenylpropanoid pathway. Accordingly, the anthocyanin content of Populus overexpressing PtoMYB10 decreased by more than 68%. These results indicate that PtoMYB10 can positively regulate xylary fiber SCW thickening, accompanied by the reprogramming of phenylpropanoid metabolism, which redirects metabolic flux from anthocyanin biosynthesis to monolignol biosynthesis. • OE- PtoMYB10 induces SCW thickening and ectopic deposition of lignin. • OE- PtoMYB10 promotes lignin biosynthesis gene expression. • OE- PtoMYB10 represses the anthocyanin accumulation. • OE- PtoMYB10 reprograms phenylpropanoid biosynthesis by redirecting metabolic flux. • Functional diversity between PtoMYB10 and its Arabidopsis counterpart. [ABSTRACT FROM AUTHOR]

Apple is a major fruit crop grown worldwide and provides humans with an essential diet and health benefits. One of the health benefits is related to the accumulation of fruit anthocyanin, which also provides fruit with an attractive red colour. It is known that an MdMYB10 allele containing a transposable element (TE) insertion in its promoter (termed Red-TE allele) underlies anthocyanin accumulation and red colouration in the fruit skin of cultivated apples. However, the distribution of this Red-TE allele in wider Malus germplasm accessions is not clear. In this study, we showed that MdMYB10 RNA in fruit skin was specifically expressed from the Red-TE allele by using allele-specific expression analysis of transcriptome data. Apple cultivars and hybrids with homozygous Red-TE alleles showed stronger red colour than those with heterozygous alleles after analysing 65 cultivars and 337 hybrids. Furthermore, both hetero- and homozygous plants growing in the same high-temperature conditions had different colourations. However, the Red-TE allele was not detected in 16 wild apple accessions showing red skin, indicating that the red skin colour of these wild apples was not conferred by the Red-TE allele. These findings provide guidance for selecting cultivars able to develop consistent red colouration under high growth temperature conditions and open the opportunity for identifying novel genetic variants underpinning fruit red colouration in wild apple species.

The red to blue hue of plant organs is caused due to anthocyanins, which are water-soluble flavonoid pigments. The accumulation of these pigments is regulated by a complex of R2R3-MYB transcription factors (TFs), basic-helix-loop-helix (bHLH), and WD-repeat (WDR) proteins (MBW complex). In Rosaceae species, R2R3-MYBs, particularly MYB10 genes, are responsible for part of the natural variation in anthocyanin colors. Japanese plum cultivars, which are hybrids of Prunus salicina, have high variability in the color hue and pattern, going from yellow-green to red and purple-blue, probably as a result of the interspecific hybridization origin of the crop. Because of such variability, Japanese plum can be considered as an excellent model to study the color determination in Rosaceae fruit tree species. Here, we cloned and characterized the alleles of the PsMYB10 genes in the linkage group LG3 region where quantitative trait loci (QTLs) for the organ color have been mapped to other Prunus species. Allele segregation in biparental populations as well as in a panel of varieties, combined with the whole-genome sequence of two varieties with contrasting fruit color, allowed the organization of the MYB10 alleles into haplotypes. With the help of this strategy, alleles were assigned to genes and at least three copies of PsMYB10.1 were identified in some varieties. In total, we observed six haplotypes, which were able to characterize 91.36% of the cultivars. In addition, two alleles of PsMYB10.1 were found to be highly associated with anthocyanin and anthocyanin-less skin. Their expression during the fruit development confirms their role in the fruit skin coloration. Here, we provide a highly efficient molecular marker for the early selection of colored or non-colored fruits in Japanese plum breeding programs.

The red to blue hue of plant organs is caused due to anthocyanins, which are water-soluble flavonoid pigments. The accumulation of these pigments is regulated by a complex of R2R3-MYB transcription factors (TFs), basic-helix-loop-helix (bHLH), and WD-repeat (WDR) proteins (MBW complex). In Rosaceae species, R2R3-MYBs, particularly MYB10 genes, are responsible for part of the natural variation in anthocyanin colors. Japanese plum cultivars, which are hybrids of Prunus salicina , have high variability in the color hue and pattern, going from yellow-green to red and purple-blue, probably as a result of the interspecific hybridization origin of the crop. Because of such variability, Japanese plum can be considered as an excellent model to study the color determination in Rosaceae fruit tree species. Here, we cloned and characterized the alleles of the PsMYB10 genes in the linkage group LG3 region where quantitative trait loci (QTLs) for the organ color have been mapped to other Prunus species. Allele segregation in biparental populations as well as in a panel of varieties, combined with the whole-genome sequence of two varieties with contrasting fruit color, allowed the organization of the MYB10 alleles into haplotypes. With the help of this strategy, alleles were assigned to genes and at least three copies of PsMYB10.1 were identified in some varieties. In total, we observed six haplotypes, which were able to characterize 91.36% of the cultivars. In addition, two alleles of PsMYB10.1 were found to be highly associated with anthocyanin and anthocyanin-less skin. Their expression during the fruit development confirms their role in the fruit skin coloration. Here, we provide a highly efficient molecular marker for the early selection of colored or non-colored fruits in Japanese plum breeding programs. [ABSTRACT FROM AUTHOR]

The flower color of many horticultural plants fades from red to white during the development stages, affecting ornamental value. We selected Malus halliana, a popular ornamental species, and analyzed the mechanisms of flower color fading using RNA sequencing. Forty-seven genes related to anthocyanin biosynthesis and two genes related to anthocyanin transport were identified; the expression of most of these genes declined dramatically with flower color fading, consistent with the change in the anthocyanin content. A number of transcription factors that might participate in anthocyanin biosynthesis were selected and analyzed. A phylogenetic tree was used to identify the key transcription factor. Using this approach, we identified MhMYB10 as directly regulating anthocyanin biosynthesis. MhMYB10 expression was strongly downregulated during flower development and was significantly positively related to the expression of anthocyanin biosynthetic genes and anthocyanin content in diverse varieties of Malus. To analyze the methylation level during flower development, the MhMYB10 promoter sequence was divided into 12 regions. The methylation levels of the R2 and R8 increased significantly as flower color faded and were inversely related to MhMYB10 expression and anthocyanin content. Therefore, we deduce that the increasing methylation activities of these two regions repressed MhMYB10 expression.

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The flower color of many horticultural plants fades from red to white during the development stages, affecting ornamental value. We selected Malus halliana , a popular ornamental species, and analyzed the mechanisms of flower color fading using RNA sequencing. Forty-seven genes related to anthocyanin biosynthesis and two genes related to anthocyanin transport were identified; the expression of most of these genes declined dramatically with flower color fading, consistent with the change in the anthocyanin content. A number of transcription factors that might participate in anthocyanin biosynthesis were selected and analyzed. A phylogenetic tree was used to identify the key transcription factor. Using this approach, we identified MhMYB10 as directly regulating anthocyanin biosynthesis. MhMYB10 expression was strongly downregulated during flower development and was significantly positively related to the expression of anthocyanin biosynthetic genes and anthocyanin content in diverse varieties of Malus. To analyze the methylation level during flower development, the MhMYB10 promoter sequence was divided into 12 regions. The methylation levels of the R2 and R8 increased significantly as flower color faded and were inversely related to MhMYB10 expression and anthocyanin content. Therefore, we deduce that the increasing methylation activities of these two regions repressed MhMYB10 expression. [ABSTRACT FROM AUTHOR]

'Yunhongli No. 1' is a rare and well-colored red pear (Pyrus pyrifolia) germplasm resource, and is popular in the market due to its bright red color and high quality. Light induces the expression of transportation factor genes MYB10 , WD40 , and HY5 , which then activate the expression of critical genes in the anthocyanin biosynthesis pathway to promote the synthesis and accumulation of anthocyanin, thus giving the red coloration. Protein HY5 is considered to be a key regulator for induction of anthocyanin biosynthesis. The MYB10 genes physically interact with HY5 to positively regulate anthocyanin biosynthesis in Arabidopsis, apple, and pear by binding to G-box motifs. However, how these transcription factors are regulated by sunlight remains unclear in 'Yunhongli No. 1'. In this study, the transcription factor PyHY5 was cloned, and subcellular localization assay showed that PyHY5 was distributed in the nucleus. The DNA fragments of PyHY5 had a typical BRLZ domain of the bZIP family, and then were aligned against the promoter sequences of PyMYB10 and PyWD40. Electrophoretic mobility shift and transient expression assays showed that PyHY5 could directly recognize and bind to the G-box motifs in the promoters of PyMYB10 and PyWD40 , and so boosted transcriptional activation by co-expression. The results demonstrated that PyHY5 binding to G-box motifs of the promoters of PyMYB10 and PyWD40 , enhanced its expression, and then promoted accumulation of anthocyanin in red 'Yunhongli No. 1'. • 'Yunhongli No. 1′ is a rare red pear with bright color. • The transcription factors PyHY5 variant in 'Yunhongli No. 1′. • The PyHY5 with a bZIP domain can also realize the same function in other plants, namely anthocyanin accumulation. • PyHY5 binding to the promoters of PyMYB10 and PyWD40 regulates the peel color of red pear. [ABSTRACT FROM AUTHOR]

Fragaria nilgerrensis is a wild diploid strawberry species endemic to east and southeast region in Asia and provides a rich source of genetic variations for strawberry improvement. Here, we present a chromosome‐scale assembly of F. nilgerrensis using single‐molecule real‐time (SMRT) Pacific Biosciences sequencing and chromosome conformation capture (Hi‐C) genome scaffolding. The genome assembly size was 270.3 Mb, with a contig N50 of ∼8.5 Mb. A total of 28 780 genes and 117.2 Mb of transposable elements were annotated for this genome. Next, detailed comparative genomics with the high‐quality F. vesca reference genome was conducted to obtain the difference among transposable elements, SNPs, Indels, and so on. The genome size of F. nilgerrensis was enhanced by around 50 Mb relatively to F. vesca, which is mainly due to expansion of transposable elements. In comparison with the F. vesca genome, we identified 4 561 825 SNPs, 846 301 Indels, 4243 inversions, 35 498 translocations and 10 099 relocations. We also found a marked expansion of genes involved in phenylpropanoid biosynthesis, starch and sucrose metabolism, cyanoamino acid metabolism, plant–pathogen interaction, brassinosteroid biosynthesis and plant hormone signal transduction in F. nilgerrensis, which may account for its specific phenotypes and considerable environmental adaptability. Interestingly, we found sequence variations in the upstream regulatory region of FnMYB10, a core transcriptional activator of anthocyanin biosynthesis, resulted in the low expression level of the FnMYB10 gene, which is likely responsible for white fruit phenotype of F. nilgerrensis. The high‐quality F. nilgerrensis genome will be a valuable resource for biological research and comparative genomics research. [ABSTRACT FROM AUTHOR]

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel‐coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red‐fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6‐phosphate. RNA‐Seq analysis showed down‐regulation of both gene‐encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6‐phosphate synthase gene TPS1.1a was down‐regulated, whereas putative regulatory TPS7 and TPS11 were strongly up‐regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up‐regulated, whereas MYB27 was also up‐regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6‐phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits. In carbon‐starved red‐fleshed kiwifruit, both carbohydrate and anthocyanin concentrations were greatly reduced. Our model suggests that anthocyanin biosynthesis was actively repressed by MYB27 via trehalose 6‐phosphate signalling to conserve carbon. [ABSTRACT FROM AUTHOR]

Summary: Light is indispensable for the accumulation of anthocyanin in the peel of red pear fruit (Pyrus pyrifolia Nakai). ELONGATED HYPOCOTYL 5 (HY5) is considered to be a critical regulator for induction of anthocyanin biosynthesis, but detailed characterization of its regulatory mechanism is needed. In this study, multiple genetic and biochemical approaches were applied to identify the roles of P. pyrifolia HY5 (PpHY5) and two B‐box (BBX) proteins, PpBBX18 and PpBBX21, in the transcriptional regulation of PpMYB10. The functions of the two BBX proteins were analyzed in overexpression lines using pear calli‐based approaches. On its own PpHY5 was unable to activate downstream genes. The two BBX proteins, PpBBX18 and PpBBX21, physically interacted with PpHY5 and antagonistically regulated anthocyanin biosynthesis in Arabidopsis and pear. PpBBX18 formed a heterodimer with PpHY5 via two B‐box domains, in which PpHY5 bound to the G‐box motif of PpMYB10 and PpBBX18 provided the trans‐acting activity, thus inducing transcription of PpMYB10. PpBBX21 interacted with PpHY5 and PpBBX18 and hampered formation of the PpHY5–PpBBX18 active transcription activator complex, and subsequently repressed anthocyanin biosynthesis. The present results demonstrate the fine‐tuned regulation of anthocyanin biosynthesis via transcriptional regulation of PpMYB10 by PpHY5‐associated proteins and provide insights into light‐induced anthocyanin biosynthesis. Significance Statement: Light is required for anthocyanin biosynthesis in red pears, and PpMYB10 plays an important role in the light‐induced anthocyanin accumulation pathway. In this study, we proved that the key factor responding to light signals, Pyrus pyrifolia ELONGATED HYPOCOTYL 5, and two B‐box proteins, PpBBX18 and PpBBX21, coordinately regulate transcription of PpMYB10. Our findings illustrate the complex transcriptional regulatory network modulating anthocyanin accumulation in anthocyanin biosynthesis in fruit. [ABSTRACT FROM AUTHOR]

Summary: The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

Carotenoids are essential and beneficial substances for both plant and human health. Identifying the regulatory network of these pigments is necessary for improving fruit quality and commodity value. In this study, we performed integrative analyses of transcriptome data from two different type fruits, ripening peel color at green ('Neelum' mango) and red ('Irwin' mango). Specifically, we found that MiMYB10 transcription level was highly associated with mango peel color. Further, silencing MiMYB10 homologous gene in tomato fruits resulted in lower carotenoid and anthocyanin content. Electrophoretic mobility shift assays and dual-luciferase clarified that MiMYB10 regulates the carotenoid biosynthesis gene MiPDS (phytoene desaturase gene) in a direct manner. On the other hand, MiMYB10 activates the expression of carotenoid biosynthesis genes (PSY , Z-ISO , CRTISO , LCYE) and chlorophyll degradation gene (SGR1), promoting the accumulation of carotenoid, accelerating chlorophyll degradation, and controlling peel color. In summary, this study identified important roles of MiMYB10 in pigment regulatory and provided new options for breeding strategies aiming to improve fruit quality. [ABSTRACT FROM AUTHOR]

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems.

Most red-fleshed kiwifruit cultivars, such as Hongyang, only accumulate anthocyanins in the inner pericarp; the trait of full red flesh becomes the goal pursued by breeders. In this study, we identified a mutant "H-16" showing a red color in both the inner and outer pericarps, and the underlying mechanism was explored. Through transcriptome analysis, a key differentially expressed gene AcGST1 was screened out, which was positively correlated with anthocyanin accumulation in the outer pericarp. The result of McrBC-PCR and bisulfite sequencing revealed that the SG3 region (-292 to -597 bp) of AcGST1 promoter in "H-16" had a significantly lower CHH cytosine methylation level than that in Hongyang, accompanied by low expression of methyltransferase genes ( MET1 and CMT2 ) and high expression of demethylase genes ( ROS1 and DML1 ). Transient calli transformation confirmed that demethylase gene DML1 can activate transcription of AcGST1 to enhance its expression. Overexpression of AcGST1 enhanced the anthocyanin accumulation in the fruit flesh and leaves of the transgenic lines. These results suggested that a decrease in the methylation level of the AcGST1 promoter may contribute to accumulation of anthocyanin in the outer pericarp of "H-16".

The influence of colored plastic films (red, yellow, green, blue, and white) on fruit quality, antioxidant capacity, and gene transcripts was studied in greenhousegrown strawberries. Fruits grown under white plastic film were used as the controls. Results indicated that there was no difference in single fruit weight due to colored plastic films in the present study. The colored plastic films had significant effects on sugar and organic acid content. The content of total sugar (SUG) was increased by 10.39% and total organic acid (ACID) was decreased by 16.58% in fruit grown under blue plastic film compared with the controls. Fruit grown under blue plastic film had significantly higher SUG content and lower ACID content than fruit subjected to yellow and green plastic films and had the highest SUG/ACID ratio of 11.46. Colored plastic films had significant effects on bioactive compound (anthocyanin, flavonoid, phenolic) content and antioxidant capacity. The highest level of bioactive compound content was detected under red plastic film. The content of total phenolics, total flavonoids, and total anthocyanin (TAC) in fruits grown under red plastic film was respectively 23.10%, 25.37%, and 74.11% higher than that of the fruits grown under the control. The antioxidant capacities were highest in fruits grown under red plastic film. Fruits grown under red and yellow films had higher sucrose phosphate synthase (SPS) and sucrose synthase (SS) activity than those fruit covered with green, blue, and white plastic films at whole fruit development stages. Acid invertase (AI) activity was high in fruit grown under green plastic film and declined during development. Blue plastic film had mainly increased the FaSPS and FaAI transcript at the green stage, and decreased the expression of FaSS. It is proposed that colored plastic films can regulate the expression of genes involved in the flavonoid biosynthesis pathway, especially FaPAL, FaF3H, FaFGT, and FaMYB10, at half-red and red stages. [ABSTRACT FROM AUTHOR]

Blush, the proportion of red overcolor on the skin surface of fruit, is highly variable in peach breeding germplasm and is important in the marketing of peach fruit. The fresh market peach industry demands a high level of blush to entice consumers, while the processing peach industry requires minimal blush. Therefore, blush is a major selection criterion in breeding programs. The use of DNA-based information could improve breeding efficiency and accuracy for fruit blush coverage, but a predictive DNA test is required. The objective of this study was to develop a DNA test for the prediction of blush coverage by targeting the major locus, R , associated with blush variation. Initially, haplotypes were developed based on five SNP markers associated with variation in blush coverage. To convert the 5-SNP haplotype test into a single, simple PCR-based assay, 11 simple sequence repeat markers were designed and used to screen individuals representing all SNP haplotypes. The most informative assay, named Ppe-R-SSR, was chosen to screen 200 individuals of the RosBREED peach reference germplasm set that incorporated germplasm from four breeding programs. Ppe-R-SSR accurately differentiated individuals with high-, medium-, and low-blush coverage in most lineages. Outcomes highlighted that DNA tests can be quite predictive for some breeding programs or specific germplasm sets, while for others the predictiveness can falter. Therefore, the confirmation of genotype effects for any DNA test is recommended in new germplasm before routine use. The prediction accuracy and breeding utility of Ppe-R-SSR in the University of Arkansas breeding program were subsequently confirmed by screening 443 seedlings, independent of the initial DNA test development process, derived from 18 cross-combinations of 28 parents. Ppe-R-SSR can be used to efficiently and accurately predict fruit blush coverage, especially in fresh market germplasm, and has been deployed for routine use in the University of Arkansas peach breeding program. [ABSTRACT FROM AUTHOR]

Sweet cherry fruit color is a market class-defining trait. The two main market classes in the USA are mahogany, consisting fruit with red skin and flesh, and blush, consisting clear-fleshed fruit with yellow skin and a red overcolor on less than the entire skin surface. Fruit color is a major consideration in sweet cherry breeding as resources and selection thresholds are often differentially applied to each market class. The use of DNA-based information could improve breeding efficiency and accuracy for fruit color, but a predictive DNA test is required. The objective of this study was to develop a reliable, simple DNA test for the prediction of sweet cherry color-based market classes, targeting the major locus, termed here as Rf, associated with fruit color variation. Haplotypes were developed based on 14 SNP markers from the RosBREED cherry 6K SNP array v1 that were associated with the two market classes. To convert the multiple SNP markers to a single, simple PCR-based assay, 11 PCR-based assays targeting microsatellite motifs were designed, using the peach reference genome sequence, and used to screen 20 individuals representing the most common SNP haplotypes. One assay, subsequently named Pav-Rf-SSR, was used to screen 221 phenotyped individuals of the RosBREED sweet cherry reference germplasm set and accurately differentiated individuals with mahogany and blush fruits. Pav-Rf-SSR can be used in DNA-informed breeding schemes to efficiently and accurately predict genetic potential for fruit color and is one of the first DNA tests publicly available for a sweet cherry fruit quality trait. [ABSTRACT FROM AUTHOR]

Anthocyanins are flavonoid pigments imparting red, blue, or purple pigmentation to fruits, flowers and foliage. These compounds are powerful antioxidants in vitro, and are widely believed to contribute to human health. The fruit of the domestic apple ( Malus x domestica) is a popular and important source of nutrients, and is considered one of the top 'functional foods'-those foods that have inherent health-promoting benefits beyond basic nutritional value. The pigmentation of typical red apple fruits results from accumulation of anthocyanin in the skin. However, numerous genotypes of Malus are known that synthesize anthocyanin in additional fruit tissues including the core and cortex (flesh). Red-fleshed apple genotypes are an attractive starting point for development of novel varieties for consumption and nutraceutical use through traditional breeding and biotechnology. However, cultivar development is limited by lack of characterization of the diversity of genetic backgrounds showing this trait. We identified and cataloged red-fleshed apple genotypes from four Malus diversity collections representing over 3,000 accessions including domestic cultivars, wild species, and named hybrids. We found a striking range of flesh color intensity and pattern among accessions, including those carrying the MYB10 R allele conferring ectopic expression of a key transcriptional regulator of anthocyanin biosynthesis. Although MYB10 R was strongly associated with red-fleshed fruit among genotypes, this allele was neither sufficient nor required for this trait in all genotypes. Nearly all red-fleshed accessions tested could be traced back to 'Niedzwetzkyana', a presumed natural form of M. sieversii native to central Asia. [ABSTRACT FROM AUTHOR]

* Premise of the study: Duchesnea indica is a wild strawberry-like species that has red fruits. In a recent survey in the highlands of Tucumán (Argentina), a plant of D. indica with white fruits was discovered. The aim of this study was to investigate whether the white-fruited character was due to a phenotypic or genotypic change. The stability and heritability of the character and the expression of genes involved in anthocyanins synthesis were studied and compared with red-fruited genotypes. This study contributes to understanding the molecular basis of some factors involved in fruit pigmentation, a horticulturally and taxonomically important trait. * Methods: Stability and heritability of the white-fruited character were evaluated in plants obtained by asexual propagation or by sexual crosses between the white- and red-fruited genotypes. Asexual multiplications were carried out by stolon rooting and sexual multiplications by germination of achenes obtained from crosses. The expression level of the genes involved in the synthesis and regulation of the anthocyanins pathway (CHS , F3H , DFR , ANS, and MYB10) were evaluated by RT-PCR using specific primers. * Key results: Plants with the white-fruited character always yielded white-fruited progeny when propagated asexually, whereas in sexually propagated plants fruit color depended on the mother. Red-fruited mothers yielded red-fruited progeny, and white-fruited mothers yielded fruits ranging from dark pink to white. Molecular analysis suggested that the white-fruited character was due to the low expression of the ANS gene. * Conclusions: Results obtained indicate that the white-fruited character was stable. Mother progenitors exert a strong influence on the expression of the white-fruited character. The white-fruited phenotype is due to the impairment or downregulation of the ANS gene. [ABSTRACT FROM AUTHOR]

The genus Fragaria encompass fruits with diverse colors influenced by the distribution and accumulation of anthocyanin. Particularly, the fruit colors of strawberries with different ploidy levels are determined by expression and natural variations in the vital structural and regulatory genes involved in the anthocyanin pathway. Among the regulatory genes, MYB10 transcription factor is crucial for the expression of structural genes in the anthocyanin pathway. In the present study, we performed a genome wide investigation of MYB10 in the diploid and octoploid Fragaria species. Further, we identified seven quantitative trait loci (QTLs) associated with fruit color in octoploid strawberry. In addition, we predicted 20 candidate genes primarily influencing the fruit color based on the QTL results and transcriptome analysis of fruit skin and flesh tissues of light pink, red, and dark red strawberries. Moreover, the computational and transcriptome analysis of MYB10 in octoploid strawberry suggests that the difference in fruit colors could be predominantly influenced by the expression of MYB10 from the F. iinumae subgenome. The outcomes of the present endeavor will provide a platform for the understanding and tailoring of anthocyanin pathway in strawberry for the production of fruits with aesthetic colors. [ABSTRACT FROM AUTHOR]

Analysis of codon usage bias (CUB) in different species can reveal the patterns of genetic information transfer across those species. To better understand the characteristics of MYB10 —a key regulator of anthocyanin biosynthesis—and identify the true (functional) MYB10 gene among the two candidates in Populus , we analysed the coding sequences of MYB10 genes in 10 different species using Codon W, CHIPS, CUSP, and CAI. Majority of the optimal amino acid codons of MYB10 genes ended with A/U, and GGA, UCA, GCA, AGA, and CCA were over-represented in all plant species studied. Among the two most promising MYB10 gene candidates in Populus , Potri.17G125700 shared a higher similarity of codon usage with MYB10 genes from other plant species, suggesting that it encodes the functional MYB10 in Populus. We verified this speculation by cloning both candidate MYB10 genes from Populus into vectors to produce transiently transformed seedlings. Colour phenotypes and anthocyanin content of the transiently transformed seedlings indicated that Potri.17G125700 encodes the true MYB10 transcription factor, which positively regulates anthocyanin accumulation in Populus. Furthermore, CUB analysis was used to select the most promising MYB12 candidate in Malus sp. (crabapple). Our results demonstrate the effectiveness of CUB analysis as a promising method to identify the functional gene from a set of candidates in long-living plants with complex genetics. [ABSTRACT FROM AUTHOR]

This study aims to investigate the molecular mechanism of the transcription factor MYB10, which is involved in anthocyanin biosynthesis, in different colors of Ribes L. fruitification. Rapid amplification of cDNA ends (RACE) was used to clone the MYB10 genes from Ribes nigrum L. ( RnMYB10 ), Ribes rubrum L. ( RrMYB10 ), and Ribes album L. ( RaMYB10 ), respectively. Phylogenetic analysis showed that RnMYB10 and RrMYB10 were evolutionarily homologous. Real-time quantitative PCR (RT-qPCR) showed that the expression of MYB10 in the fruits of Ribes nigrum L. was higher than that of Ribes rubrum L. and much higher than that of Ribes album L. The expression of RnMYB10 and RrMYB10 increased at first and then decreased as the fruit diameter increased and the fruit color deepened (the maximum expression level was reached at 75% of the fruit color change), while the expression level of RaMYB10 was very low. Overexpression of RnMYB10 and RrMYB10 in Arabidopsis thaliana resulted in purple petioles and leaves, whereas overexpression of RaMYB10 resulted in no significant color changes. This indicates that MYB10 gene plays an important role in the coloration of Ribes L. fruit.

Background: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. Results: We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologies Cleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. Conclusions: We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches. [ABSTRACT FROM AUTHOR]

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems. [ABSTRACT FROM AUTHOR]