Identification of miRNA858 long-loop precursors in seed plants.

MicroRNAs (miRNAs) are a class of nonprotein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this subclass. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100 to 2,100 nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350 and 5,500 nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.

The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus.

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in the premature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAs and reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plant height.

L-Ascorbic acid metabolism and regulation in fruit crops.

L-Ascorbic acid (AsA) is more commonly known as vitamin C and is an indispensable compound for human health. As a major antioxidant, AsA not only maintains redox balance and resists biological and abiotic stress but also regulates plant growth, induces flowering, and delays senescence through complex signal transduction networks. However, AsA content varies greatly in horticultural crops, especially in fruit crops. The AsA content of the highest species is approximately 1,800 times higher than that of the lowest species. There have been significant advancements in the understanding of AsA accumulation in the past 20 years. The most noteworthy accomplishment was the identification of the critical rate-limiting genes for the 2 major AsA synthesis pathways (L-galactose pathway and D-galacturonic acid pathway) in fruit crops. The rate-limiting genes of the former are GMP, GME, GGP, and GPP, and the rate-limiting gene of the latter is GalUR. Moreover, APX, MDHAR, and DHAR are also regarded as key genes in degradation and regeneration pathways. Interestingly, some of these key genes are sensitive to environmental factors, such as GGP being induced by light. The efficiency of enhancing AsA content is high by editing upstream open reading frames (uORF) of the key genes and constructing multi-gene expression vectors. In summary, the AsA metabolism has been well understood in fruit crops, but the transport mechanism of AsA and the synergistic improvement of AsA and other traits is less known, which will be the focus of AsA research in fruit crops.

Colorful hues: insight into the mechanisms of anthocyanin pigmentation in fruit.

Anthocyanin is a vital indicator for both fruit nutritional and commercial value. Anthocyanin accumulation is a surprisingly complicated process mediated by multiple networks associated with genetic, developmental, hormonal, and environmental factors. Transcriptional regulation along with epigenetic regulation constitutes the dominant molecular framework for anthocyanin biosynthesis. Here, we focus on current knowledge on regulatory mechanisms of anthocyanin accumulation, with emphasis on the latest progress in transcriptional and epigenetic regulation and the crosstalk between various signaling pathways. We present an emerging picture of how various internal and external stimuli control anthocyanin biosynthesis. Additionally, we discuss the synergistic or antagonistic effect of developmental, hormonal and environmental cues on anthocyanin accumulation in fruit.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

DNA methylation controlling abscisic acid catabolism responds to light to mediate strawberry fruit ripening.

Phytohormones, epigenetic regulation and environmental factors regulate fruit ripening but their interplay during strawberry fruit ripening remains to be determined. In this study, bagged strawberry fruit exhibited delayed ripening compared with fruit grown in normal light, correlating with reduced abscisic acid (ABA) accumulation. Transcription of the key ABA catabolism gene, ABA 8'-hydroxylase FaCYP707A4, was induced in bagged fruit. With light exclusion whole genome DNA methylation levels were up-regulated, corresponding to a delayed ripening process, while DNA methylation levels in the promoter of FaCYP707A4 were suppressed, correlating with increases in transcript and decreased ABA content. Experiments indicated FaCRY1, a blue light receptor repressed in bagged fruit and FaAGO4, a key protein involved in RNA-directed DNA methylation, could bind to the promoter of FaCYP707A4. The interaction between FaCRY1 and FaAGO4, and an increased enrichment of FaAGO4 directed to the FaCYP707A4 promoter in fruit grown under light suggests FaCRY1 may influence FaAGO4 to modulate the DNA methylation status of the FaCYP707A4 promoter. Furthermore, transient overexpression of FaCRY1, or an increase in FaCRY1 transcription by blue light treatment, increases the methylation level of the FaCYP707A4 promoter, while transient RNA interference of FaCRY1 displayed opposite phenotypes. These findings reveal a mechanism by which DNA methylation influences ABA catabolism, and participates in light-mediated strawberry ripening.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

Why do plants blush when they are hungry?

Foliar anthocyanins, as well as other secondary metabolites, accumulate transiently under nutritional stress. A misconception that only nitrogen or phosphorus deficiency induces leaf purpling/reddening has led to overuse of fertilizers that burden the environment. Here, we emphasize that several other nutritional imbalances induce anthocyanin accumulation, and nutrient-specific differences in this response have been reported for some deficiencies. A range of ecophysiological functions have been attributed to anthocyanins. We discuss the proposed functions and signalling pathways that elicit anthocyanin synthesis in nutrient-stressed leaves. Knowledge from the fields of genetics, molecular biology, ecophysiology and plant nutrition is combined to deduce how and why anthocyanins accumulate under nutritional stress. Future research to fully understand the mechanisms and nuances of foliar anthocyanin accumulation in nutrient-stressed crops could be utilized to allow these leaf pigments to act as bioindicators for demand-oriented application of fertilizers. This would benefit the environment, being timely due to the increasing impact of the climate crisis on crop performance.

Model-free portfolio theory: A rough path approach.

Based on a rough path foundation, we develop a model-free approach to stochastic portfolio theory (SPT). Our approach allows to handle significantly more general portfolios compared to previous model-free approaches based on Föllmer integration. Without the assumption of any underlying probabilistic model, we prove a pathwise formula for the relative wealth process, which reduces in the special case of functionally generated portfolios to a pathwise version of the so-called master formula of classical SPT. We show that the appropriately scaled asymptotic growth rate of a far reaching generalization of Cover's universal portfolio based on controlled paths coincides with that of the best retrospectively chosen portfolio within this class. We provide several novel results concerning rough integration, and highlight the advantages of the rough path approach by showing that (nonfunctionally generated) log-optimal portfolios in an ergodic Itô diffusion setting have the same asymptotic growth rate as Cover's universal portfolio and the best retrospectively chosen one.

CRISPR-Cas9-mediated mutagenesis of kiwifruit BFT genes results in an evergrowing but not early flowering phenotype.

Phosphatidylethanolamine-binding protein (PEBP) genes regulate flowering and architecture in many plant species. Here, we study kiwifruit (Actinidia chinensis, Ac) PEBP genes with homology to BROTHER OF FT AND TFL1 (BFT). CRISPR-Cas9 was used to target AcBFT genes in wild-type and fast-flowering kiwifruit backgrounds. The editing construct was designed to preferentially target AcBFT2, whose expression is elevated in dormant buds. Acbft lines displayed an evergrowing phenotype and increased branching, while control plants established winter dormancy. The evergrowing phenotype, encompassing delayed budset and advanced budbreak after defoliation, was identified in multiple independent lines with edits in both alleles of AcBFT2. RNA-seq analyses conducted using buds from gene-edited and control lines indicated that Acbft evergrowing plants had a transcriptome similar to that of actively growing wild-type plants, rather than dormant controls. Mutations in both alleles of AcBFT2 did not promote flowering in wild-type or affect flowering time, morphology and fertility in fast-flowering transgenic kiwifruit. In summary, editing of AcBFT2 has the potential to reduce plant dormancy with no adverse effect on flowering, giving rise to cultivars better suited for a changing climate.

A MADS-box gene with similarity to FLC is induced by cold and correlated with epigenetic changes to control budbreak in kiwifruit.

Temperate perennials require exposure to chilling temperatures to resume growth in the following spring. Growth and dormancy cycles are controlled by complex genetic regulatory networks and are governed by epigenetic mechanisms, but the specific genes and mechanisms remain poorly understood. To understand how seasonal changes and chilling regulate dormancy and growth in the woody perennial vine kiwifruit (Ac, Actinidia chinensis), a transcriptome study of kiwifruit buds in the field and controlled conditions was performed. A MADS-box gene with homology to Arabidopsis FLOWERING LOCUS C (FLC) was identified and characterized. Elevated expression of AcFLC-like (AcFLCL) was detected during bud dormancy and chilling. A long noncoding (lnc) antisense transcript with an expression pattern opposite to AcFLCL and shorter sense noncoding RNAs were identified. Chilling induced an increase in trimethylation of lysine-4 of histone H3 (H3K4me3) in the 5' end of the gene, indicating multiple layers of epigenetic regulation in response to cold. Overexpression of AcFLCL in kiwifruit gave rise to plants with earlier budbreak, whilst gene editing using CRISPR-Cas9 resulted in transgenic lines with substantially delayed budbreak, suggesting a role in activation of growth. These results have implications for the future management and breeding of perennials for resilience to changing climate.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

Hierarchical regulation of MYBPA1 by anthocyanin- and proanthocyanidin-related MYB proteins is conserved in Vaccinium species.

Members of the Vaccinium genus bear fruits rich in anthocyanins, a class of red-purple flavonoid pigments that provide human health benefits, although the localization and concentrations of anthocyanins differ between species: blueberry (V. corymbosum) has white flesh, while bilberry (V. myrtillus) has red flesh. Comparative transcriptomics between blueberry and bilberry revealed that MYBPA1.1 and MYBA1 strongly correlated with the presence of anthocyanins, but were absent or weakly expressed in blueberry flesh. MYBPA1.1 had a biphasic expression profile, correlating with both proanthocyanidin biosynthesis early during fruit development and anthocyanin biosynthesis during berry ripening. MYBPA1.1 was unable to induce anthocyanin or proanthocyanidin accumulation in Nicotiana benthamiana, but activated promoters of flavonoid biosynthesis genes. The MYBPA1.1 promoter is directly activated by MYBA1 and MYBPA2 proteins, which regulate anthocyanins and proanthocyanidins, respectively. Our findings suggest that the lack of VcMYBA1 expression in blueberry flesh results in an absence of VcMYBPA1.1 expression, which are both required for anthocyanin regulation. In contrast, VmMYBA1 is well expressed in bilberry flesh, up-regulating VmMYBPA1.1, allowing coordinated regulation of flavonoid biosynthesis genes and anthocyanin accumulation. The hierarchal model described here for Vaccinium may also occur in a wider group of plants as a means to co-regulate different branches of the flavonoid pathway.

Cortisol metabolites vary with environmental conditions, predation risk, and human shields in a wild primate, Cercopithecus albogularis.

Environmental challenges are often associated with physiological changes in wildlife that allow animals to maintain homeostasis. Among these, scarcity in resources, and risks from predators, competitors, and humans can all result in psychological and physiological stress. Yet, for habituated species, it is not clear whether this relationship with humans still holds to a lesser degree or is outweighed by the benefits of human presence - such as serving as a buffer from competitors or predators. We investigated how human presence and environmental challenges such as resource availability, weather, predation, and competition may be associated with variation in fecal cortisol metabolite levels (FCMs) in a group of samango monkeys (Cercopithecus albogularis) in the Soutpansberg Mountains, South Africa. FCMs can often broadly track environmental challenges and perturbations. Initially, we employed an exploratory analysis comparing candidate models representing biological hypotheses and found that those incorporating information on human presence had less weight than models for food availability, thermoregulation, and water scarcity. When we examined a subset of the data that included information on intergroup competition and predator alarm calls, we found that FCMs were higher on the day following potential predator encounters but not competitive interactions. As observer numbers increased, responses to predators flattened, indicating that the presence of several humans might deter predators and/or affect samangos' perception of danger - yet we could not distinguish between these possibilities. Together, these results suggest that ecological perturbations track with FCMs in this study population and challenge long-held assumptions that human presence has negligible effects on habituated study animals.

DNA demethylation is involved in the regulation of temperature-dependent anthocyanin accumulation in peach.

Anthocyanin biosynthesis is induced by low temperatures in a number of plants. However, in peach (cv Zhonghuashoutao), anthocyanin accumulation was observed in fruit stored at 16°C but not at or below 12°C. Fruit stored at 16°C showed elevated transcript levels of genes encoding anthocyanin biosynthetic enzymes, the transport protein glutathione S-transferase and key transcription factors. Higher transcript levels of PpPAL1/2, PpC4H, Pp4CL4/5/8, PpF3H, PpF3'H, PpDFR1/2/3 and PpANS, as well as transcription factor gene PpbHLH3, were associated with lower methylation levels in the promoter of these genes. The DNA methylation level was further highly correlated with the expression of the DNA methyltransferase genes and DNA demethylase genes. The application of DNA methylation inhibitor 5-azacytidine induced anthocyanin accumulation in peach flesh, further implicating a critical role for DNA demethylation in regulating anthocyanin accumulation in peach flesh. Our data reveal that temperature-dependent DNA demethylation is a key factor to the post-harvest temperature-dependent anthocyanin accumulation in peach flesh.

The interaction of MYB, bHLH and WD40 transcription factors in red pear (Pyrus pyrifolia) peel.

KEY MESSAGE: Sunlight enhanced peel color and significantly up-regulated the expression of PyMYB10 and PybHLH genes. MYB-bHLH-WD40 transcriptional complex forms in the light and is involved in regulating anthocyanin accumulation in the peel. Anthocyanin is the major pigment in the peel of Yunnan red pear (Pyrus pyrifolia (Burm.) Nak.). A transcriptional activation protein complex, involving members of the transcription factor classes of MYB, bHLH and WD40, regulates anthocyanin biosynthesis. This complex was examined in the peel of red pear. In order to clarify the interaction of PyMYB10, PybHLH and PyWD40, fruit were bagged then peel samples collected 0, 3, 5, and 7 days after bag removal. Samples were used for Western blotting and protein interaction analysis. The results showed that sunlight enhanced peel color and significantly up-regulated the expression of both PyMYB10 and PybHLH genes. Co-immunoprecipitation (Co-IP) analysis showed that PybHLH interacted with PyMYB10 or PyWD40, and PyMYB10 interacted with PyWD40. Using onion cells as a model system, bimolecular fluorescence complementation (BiFC) confirmed these interactions and showed that the interaction localized to the nuclei. GST Pull down and Far-Western blotting assays demonstrated that PybHLH interacted with PyMYB10 or PyWD40, respectively, and PyMYB10 interacted with PyWD40 in vitro. In addition, EMSA assay showed that PyMYB10 can directly bind to the promoter of the gene encoding the anthocyanin biosynthesis enzyme anthocyanidin synthase (PyANS). Taken together, these results showed that the ternary complex of PyMYB10, PybHLH and PyWD40 transcription factors forms to regulate anthocyanin biosynthesis and accumulation in Yunnan red pear.

NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

BACKGROUND: Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. RESULTS: In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. CONCLUSIONS: Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

Regulation of wound ethylene biosynthesis by NAC transcription factors in kiwifruit.

BACKGROUND: The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. RESULTS: A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1-4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. CONCLUSIONS: Our results indicate that in kiwifruit the NAC TFs AcNAC2-4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.