Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

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.

RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees.

DORMANCY-ASSOCIATED MADS-box (DAM) and SHORT VEGETATIVE PHASE (SVP) genes have been implicated in the regulation of winter dormancy in perennials. Ectopic expression of apple (Malus × domestica Borkh. 'Royal Gala') DAM and SVP genes delays budbreak and constrains lateral shoot outgrowth. In this study, we used RNA interference (RNAi) to simultaneously target all apple DAM and SVP genes in order to study their role and mode of action in the regulation of bud dormancy, budbreak and flowering. A synthetic construct carrying a hairpin fragment assembled from sequences specific to coding regions of three DAM and two SVP genes was used to generate transgenic lines. Reduced expression of DAM/SVP genes resulted in delayed leaf senescence and abscission in autumn, failure to enter bud dormancy in winter and continual growth of new leaves regardless of the season for over 3 years. Precocious flowering but normal flower morphology, fertility and fruit development were observed. The non-dormant phenotype was associated with modified phytohormone composition. The content of gibberellins (GAs) and jasmonates (JAs) was significantly increased in terminal buds of RNAi lines compared with wildtype plants, accompanied by elevated expression of the key GA biosynthesis pathway gene GIBBERELLIN 20 OXIDASE-2 (MdGA20ox-2) along with the FLOWERING LOCUS T gene MdFT2. The key mediator of plasmodesmatal closure, MdCALLOSE SYNTHASE 1 (MdCALS1), was repressed in RNAi lines. This study provides functional evidence for the role of DAM/SVP genes in vegetative phenology of apple and paves the way for production of low-chill varieties suitable for growth in warming climates.

Histone modification and activation by SOC1-like and drought stress-related transcription factors may regulate AcSVP2 expression during kiwifruit winter dormancy.

The SHORT VEGETATIVE PHASE (SVP)-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been shown to regulate winter dormancy in woody perennials. In kiwifruit, AcSVP2 affects the duration of dormancy in cultivars that require high chill for dormancy release. In this study, we used a low-chill kiwifruit Actinidia chinensis 'Hort16A' to further study the function and regulation of AcSVP2. Overexpression of AcSVP2 in transgenic A. chinensis delayed budbreak in spring. A reduction in the active trimethylation histone marks of the histone H3K4 and acetylation of histone H3 contributed to the reduction of AcSVP2 expression towards dormancy release, while the inactive histone marks of trimethylation of the histone H3K27 and H3K9 in AcSVP2 locus did not show significant enrichment at the end of winter dormancy. Analysis of expression in shoot buds showed that AcSVP2 transcript was elevated in dormant buds during winter months and declined prior to budbreak, which was coordinated with expression of some of kiwifruit SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)-like genes. Screening of 101 transcription factors in an assay with a 2.3 kb promoter region of AcSVP2 found that kiwifruit SOC1-like genes are able to activate the AcSVP2 promoter. We further identified additional transcription factors associated with drought/osmotic stress and dormancy which may regulate AcSVP2 expression.

Kiwifruit SVP2 controls developmental and drought-stress pathways.

KEY MESSAGE: Genome-wide targets of Actinidia chinensis SVP2 confirm roles in ABA- and dehydration-mediated growth repression and reveal a conservation in mechanism of action between SVP genes of taxonomically distant Arabidopsis and a woody perennial kiwifruit. The molecular mechanisms underlying growth and dormancy in woody perennials are largely unknown. In Arabidopsis, the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a key role in the progression from vegetative to floral development, and in woody perennials SVP-like genes are also proposed to be involved in controlling dormancy. During kiwifruit development SVP2 has a role in growth inhibition, with high-chill kiwifruit Actinidia deliciosa transgenic lines overexpressing SVP2 showing suppressed bud outgrowth. Transcriptomic analyses of these plants suggests that SVP2 mimics the well-documented abscisic acid (ABA) effect on the plant dehydration response. To corroborate the growth inhibition role of SVP2 in kiwifruit development at the molecular level, we analysed the genome-wide direct targets of SVP2 using chromatin immunoprecipitation followed by high-throughput sequencing in kiwifruit A. chinensis. SVP2 was found to bind to at least 297 target sites in the kiwifruit genome, and potentially modulates 252 genes that function in a range of biological processes, especially those involved in repressing meristem activity and ABA-mediated dehydration pathways. In addition, our ChIP-seq analysis reveals remarkable conservation in mechanism of action between SVP genes of taxonomically distant plant species.

AcFT promotes kiwifruit in vitro flowering when overexpressed and Arabidopsis flowering when expressed in the vasculature under its own promoter.

Kiwifruit (Actinidia chinensis) has three FLOWERING LOCUS T (FT) genes, AcFT, AcFT1, and AcFT2, with differential expression and potentially divergent roles. AcFT was previously shown to be expressed in source leaves and induced in dormant buds by winter chilling. Here, we show that AcFT promotes flowering in A. chinensis, despite a short sequence insertion not present in other FT-like genes. A 3.5-kb AcFT promoter region contained all the regulatory elements required to mediate vascular expression in transgenic Arabidopsis thaliana (Arabidopsis). The promoter activation was initially confined to the veins in the distal end of the leaf, before extending to the veins in the base of the leaf, and was detected in inductive and noninductive photoperiods. The 3-kb and 2.7-kb promoter regions of AcFT1 and AcFT2, respectively, demonstrated different activation patterns in Arabidopsis, corresponding to differential expression in kiwifruit. Expression of AcFT cDNA from the AcFT promoter was capable to induce early flowering in transgenic Arabidopsis in noninductive photoperiods. Further, expression of AcFT cDNA fused to the green fluorescent protein was detected in the vasculature and was also capable to advance flowering in noninductive photoperiods. Taken together, these studies implicate AcFT in regulation of kiwifruit flowering time and as a candidate for kiwifruit florigen.

SVP-like MADS Box Genes Control Dormancy and Budbreak in Apple.

The annual growth cycle of trees is the result of seasonal cues. The onset of winter triggers an endodormant state preventing bud growth and, once a chilling requirement is satisfied, these buds enter an ecodormant state and resume growing. MADS-box genes with similarity to Arabidopsis SHORT VEGETATIVE PHASE (SVP) [the SVP-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes] have been implicated in regulating flowering and growth-dormancy cycles in perennials. Here, we identified and characterized three DAM-like (MdDAMs) and two SHORT VEGETATIVE PHASE-like (MdSVPs) genes from apple (Malus × domestica 'Royal Gala'). The expression of MdDAMa and MdDAMc indicated they may play a role in triggering autumn growth cessation. In contrast, the expression of MdDAMb, MdSVPa and MdSVPb suggested a role in maintaining bud dormancy. Consistent with this, ectopic expression of MdDAMb and MdSVPa in 'Royal Gala' apple plants resulted in delayed budbreak and architecture change due to constrained lateral shoot outgrowth, but normal flower and fruit development. The association of MdSVPa and MdSVPb expression with floral bud development in the low fruiting 'Off' trees of a biennial bearing cultivar 'Sciros' suggested the SVP genes might also play a role in floral meristem identity.

Kiwifruit SVP2 gene prevents premature budbreak during dormancy.

Overexpression of SVP2 in kiwifruit delays budbreak before sufficient winter chilling. SVP2-mediated vegetative growth restriction involves stress response pathways, and commonalities exist between Arabidopsis and kiwifruit SVP targets.

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time.

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

Actinidia DRM1--an intrinsically disordered protein whose mRNA expression is inversely correlated with spring budbreak in kiwifruit.

Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172.

In Arabidopsis, the identity of perianth and reproductive organs are specified by antagonistic action of two floral homeotic genes, APETALA2 (AP2) and AGAMOUS (AG). AP2 is also negatively regulated by an evolutionary conserved interaction with a microRNA, miR172, and has additional roles in general plant development. A kiwifruit gene with high levels of homology to AP2 and AP2-like genes from other plant species was identified. The transcript was abundant in the kiwifruit flower, particularly petal, suggesting a role in floral organ identity. Splice variants were identified, all containing both AP2 domains, including a variant that potentially produces a shorter transcript without the miRNA172 targeting site. Increased AP2 transcript accumulation was detected in the aberrant flowers of the mutant 'Pukekohe dwarf' with multiple perianth whorls and extended petaloid features. In contrast to normal kiwifruit flowers, the aberrant flowers failed to accumulate miR172 in the developing whorls, although accumulation was detected at the base of the flower. An additional role during dormancy in kiwifruit was proposed based on AP2 transcript accumulation in axillary buds before and after budbreak.

Conservation and divergence of four kiwifruit SVP-like MADS-box genes suggest distinct roles in kiwifruit bud dormancy and flowering.

MADS-box genes similar to Arabidopsis SHORT VEGETATIVE PHASE (SVP) have been implicated in the regulation of flowering in annual species and bud dormancy in perennial species. Kiwifruit (Actinidia spp.) are woody perennial vines where bud dormancy and out-growth affect flower development. To determine the role of SVP-like genes in dormancy and flowering of kiwifruit, four MADS-box genes with homology to Arabidopsis SVP, designated SVP1, SVP2, SVP3, and SVP4, have been identified and analysed in kiwifruit and functionally characterized in Arabidopsis. Phylogenetic analysis indicate that these genes fall into different sub-clades within the SVP-like gene group, suggesting distinct functions. Expression was generally confined to vegetative tissues, and increased transcript accumulation in shoot buds over the winter period suggests a role for these genes in bud dormancy. Down-regulation before flower differentiation indicate possible roles as floral repressors. Over-expression and complementation studies in Arabidopsis resulted in a range of floral reversion phenotypes arising from interactions with Arabidopsis MADS-box proteins, but only SVP1 and SVP3 were able to complement the svp mutant. These results suggest that the kiwifruit SVP-like genes may have distinct roles during bud dormancy and flowering.

Identification and characterization of flowering genes in kiwifruit: sequence conservation and role in kiwifruit flower development.

BACKGROUND: Flower development in kiwifruit (Actinidia spp.) is initiated in the first growing season, when undifferentiated primordia are established in latent shoot buds. These primordia can differentiate into flowers in the second growing season, after the winter dormancy period and upon accumulation of adequate winter chilling. Kiwifruit is an important horticultural crop, yet little is known about the molecular regulation of flower development. RESULTS: To study kiwifruit flower development, nine MADS-box genes were identified and functionally characterized. Protein sequence alignment, phenotypes obtained upon overexpression in Arabidopsis and expression patterns suggest that the identified genes are required for floral meristem and floral organ specification. Their role during budbreak and flower development was studied. A spontaneous kiwifruit mutant was utilized to correlate the extended expression domains of these flowering genes with abnormal floral development. CONCLUSIONS: This study provides a description of flower development in kiwifruit at the molecular level. It has identified markers for flower development, and candidates for manipulation of kiwifruit growth, phase change and time of flowering. The expression in normal and aberrant flowers provided a model for kiwifruit flower development.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds.

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Ethylene-induced modulation of genes associated with the ethylene signalling pathway in ripening kiwifruit.

Gene families associated with the ethylene signal transduction pathway in ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) were isolated from a kiwifruit expressed sequence tag (EST) database, including five ethylene receptor genes, two CTR1-like genes, and an EIN3-like gene AdEIL1. All were differentially expressed among various kiwifruit vine tissues, and none was fruit specific. During fruit development, levels of transcripts of AdERS1a, AdETR3, and the two CTR1-like genes decreased, whereas those of AdERS1b and AdETR2 peaked at 97 d after full bloom. In ripening kiwifruit, there was a diverse response of the ethylene receptor family to internal and external ethylene. AdERS1a, AdETR2, and AdETR3 expression increased at the climacteric stage and transcripts were induced by external ethylene treatment, while AdERS1b showed no response to ethylene. AdETR1 was negatively regulated by internal and external ethylene in ripening fruit. The two CTR1-like genes also had different expression patterns, with AdCTR1 increasing at the climacteric stage and AdCTR2 undergoing little change. 1-Methylcyclopropene treatment prevented the ethylene response of all components, but transient down-regulation was only found with AdETR2 and AdCTR1. Similar gene and ethylene responses were found in both fruit flesh and core tissues. The ethylene-induced down-regulation of AdETR1 suggests that it may have a role in sensing ethylene and transmitting this response to other members of the receptor family, thus activating the signal transduction pathway.

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 mul of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites.

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Bimodal patterns of floral gene expression over the two seasons that kiwifruit flowers develop.

Polymerase chain reaction fragments with homology to the Arabidopsis floral meristem identity genes LEAFY and APETALA1 have been isolated from kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson) and have been named ALF and AAP1, respectively. Northern hybridisation analyses have shown that ALF and AAP1 have bimodal patterns of annual expression in developing first-order axillary buds and their subsequent shoots. This pattern of expression is consistent with the 2-year cycle of axillary bud, flower and fruit development observed in kiwifruit. The first period of expression was early in first-order bud development (late spring of the first growing season), when second-order meristems are initiated, and the second, approximately 10 months later, when those meristems differentiate flowers (late spring of the second growing season). In situ hybridisation analyses on axillary buds collected during late spring of the first growing season have shown ALF expression throughout the developing first-order buds and AAP1 expression was localised in developing second-order axillary meristems. During the spring of the second growing season, transcript accumulation for both ALF and AAP1 is localised in differentiating flowers. Our results show that important developmental events are occurring very early in kiwifruit first-order axillary bud development (spring of the first growing season) and it is likely that this includes floral commitment (evocation).