Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress.

Cold stress seriously affects plant development, resulting in heavy agricultural losses. L-ascorbic acid (AsA, vitamin C) is an antioxidant implicated in abiotic stress tolerance and metabolism of reactive oxygen species (ROS). Understanding whether and how cold stress elicits AsA biosynthesis to reduce oxidative damage is important for developing cold-resistant plants. Here, we show that the accumulation of AsA in response to cold stress is a common mechanism conserved across the plant kingdom, from single-cell algae to angiosperms. We identified a basic leucine zipper domain (bZIP) transcription factor (TF) of kiwifruit (Actinidia eriantha Benth.), AcePosF21, which was triggered by cold and is involved in the regulation of kiwifruit AsA biosynthesis and defense responses against cold stress. AcePosF21 interacted with the R2R3-MYB TF AceMYB102 and directly bound to the promoter of the gene encoding GDP-L-galactose phosphorylase 3 (AceGGP3), a key conduit for regulating AsA biosynthesis, to up-regulate AceGGP3 expression and produce more AsA, which neutralized the excess ROS induced by cold stress. On the contrary, VIGS or CRISPR-Cas9-mediated editing of AcePosF21 decreased AsA content and increased the generation of ROS in kiwifruit under cold stress. Taken together, we illustrated a model for the regulatory mechanism of AcePosF21-mediated regulation of AceGGP3 expression and AsA biosynthesis to reduce oxidative damage by cold stress, which provides valuable clues for manipulating the cold resistance of kiwifruit.

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.

A gene expression atlas for kiwifruit (Actinidia chinensis) and network analysis of transcription factors.

BACKGROUND: Transcriptomic studies combined with a well annotated genome have laid the foundations for new understanding of molecular processes. Tools which visualise gene expression patterns have further added to these resources. The manual annotation of the Actinidia chinensis (kiwifruit) genome has resulted in a high quality set of 33,044 genes. Here we investigate gene expression patterns in diverse tissues, visualised in an Electronic Fluorescent Pictograph (eFP) browser, to study the relationship of transcription factor (TF) expression using network analysis. RESULTS: Sixty-one samples covering diverse tissues at different developmental time points were selected for RNA-seq analysis and an eFP browser was generated to visualise this dataset. 2839 TFs representing 57 different classes were identified and named. Network analysis of the TF expression patterns separated TFs into 14 different modules. Two modules consisting of 237 TFs were correlated with floral bud and flower development, a further two modules containing 160 TFs were associated with fruit development and maturation. A single module of 480 TFs was associated with ethylene-induced fruit ripening. Three "hub" genes correlated with flower and fruit development consisted of a HAF-like gene central to gynoecium development, an ERF and a DOF gene. Maturing and ripening hub genes included a KNOX gene that was associated with seed maturation, and a GRAS-like TF. CONCLUSIONS: This study provides an insight into the complexity of the transcriptional control of flower and fruit development, as well as providing a new resource to the plant community. The Actinidia eFP browser is provided in an accessible format that allows researchers to download and work internally.

Shy Girl, a kiwifruit suppressor of feminization, restricts gynoecium development via regulation of cytokinin metabolism and signalling.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.

Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering.

Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)-like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9- mediated manipulation enabled functional analysis of kiwifruit CEN-like genes AcCEN4 and AcCEN. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi-allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whereas mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. CRISPR/Cas9-mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop.

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.

Three FT and multiple CEN and BFT genes regulate maturity, flowering, and vegetative phenology in kiwifruit.

Kiwifruit is a woody perennial horticultural crop, characterized by excessive vegetative vigor, prolonged juvenility, and low productivity. To understand the molecular factors controlling flowering and winter dormancy, here we identify and characterize the kiwifruit PEBP (phosphatidylethanolamine-binding protein) gene family. Five CEN-like and three BFT-like genes are differentially expressed and act as functionally conserved floral repressors, while two MFT-like genes have no impact on flowering time. FT-like genes are differentially expressed, with AcFT1 confined to shoot tip and AcFT2 to mature leaves. Both act as potent activators of flowering, but expression of AcFT2 in Arabidopsis resulted in a greater impact on plant morphology than that of AcFT1. Constitutive expression of either construct in kiwifruit promoted in vitro flowering, but AcFT2 displayed a greater flowering activation efficiency than AcFT1, leading to immediate floral transition and restriction of leaf development. Both leaf and flower differentiation were observed in AcFT1 kiwifruit lines. Sequential activation of specific PEBP genes in axillary shoot buds during growth and dormancy cycles indicated specific roles in regulation of kiwifruit vegetative and reproductive phenologies. AcCEN and AcCEN4 marked active growth, AcBFT2 was associated with suppression of latent bud growth during winter, and only AcFT was activated after cold accumulation and dormancy release.

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.

Functional diversification of the potato R2R3 MYB anthocyanin activators AN1, MYBA1, and MYB113 and their interaction with basic helix-loop-helix cofactors.

In potato (Solanum tuberosum L.), R2R3 MYBs are involved in the regulation of anthocyanin biosynthesis. We examined sequences of these MYBs in cultivated potatoes, which are more complex than diploid potato due to ploidy and heterozygosity. We found amino acid variants in the C-terminus of the MYB StAN1, termed R0, R1, and R3, due to the presence of a repeated 10-amino acid motif. These variant MYBs showed some expression in both white and pigmented tubers. We found several new alleles or gene family members of R2R3 MYBs,StMYBA1 and StMYB113, which were also expressed in white potato tubers. From functional analysis in tobacco, we showed that the presence of a C-terminal 10-amino acid motif is optimal for activating anthocyanin accumulation. Engineering a motif back into a MYB lacking this sequence enhanced its activating ability. Versions of StMYBA1 and StMYB113 can also activate anthocyanin accumulation in tobacco leaves, with the exception of StMYB113-3, which has a partial R2R3 domain. We isolated five family members of potato StbHLH1, and one StJAF13, to test their ability to interact with MYB variants. The results showed that two alleles of StbHLH1 from white skin and red skin are non-functional, while three other StbHLH1s have different co-regulating abilities, and need to be activated by StJAF13. Combined with expression analysis in potato tuber, results suggest that StbHLH1 and StJAF13a re key co-regulators of anthocyanin biosynthesis, while the transcripts of MYB variants StAN1,StMYBA1, and StMYB113 are well expressed, even in the absence of pigmentation.

Functional and expression analyses of kiwifruit SOC1-like genes suggest that they may not have a role in the transition to flowering but may affect the duration of dormancy.

The MADS-domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) is one of the key integrators of endogenous and environmental signals that promote flowering in the annual species Arabidopsis thaliana. In the deciduous woody perennial vine kiwifruit (Actinidia spp.), environmental signals are integrated to regulate annual cycles of growth and dormancy. Accumulation of chilling during winter is required for dormancy break and flowering in spring. In order to understand the regulation of dormancy and flowering in kiwifruit, nine kiwifruit SOC1-like genes were identified and characterized. All genes affected flowering time of A. thaliana Col-0 and were able to rescue the late flowering phenotype of the soc1-2 mutant when ectopically expressed. A differential capacity for homodimerization was observed, but all proteins were capable of strong interactions with SHORT VEGETATIVE PHASE (SVP) MADS-domain proteins. Largely overlapping spatial domains but distinct expression profiles in buds were identified between the SOC1-like gene family members. Ectopic expression of AcSOC1e, AcSOC1i, and AcSOC1f in Actinidia chinensis had no impact on establishment of winter dormancy and failed to induce precocious flowering, but AcSOC1i reduced the duration of dormancy in the absence of winter chilling. These findings add to our understanding of the SOC1-like gene family and the potential diversification of SOC1 function in woody perennials.

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.

SmuMYB113 is the determinant of fruit color in pepino (Solanum muricatum).

Pepino (Solanum muricatum) is an herbaceous crop phylogenetically related to tomato and potato. Pepino fruit vary in color, size and shape, and are eaten fresh. In this study, we use pepino as a fruit model to understand the transcriptional regulatory mechanisms controlling fruit quality. To identify the key genes involved in anthocyanin biosynthesis in pepino, two genotypes were studied that contrasted in foliar and fruit pigmentation. Anthocyanin profiles were analyzed, as well as the expression of genes that encode enzymes for anthocyanin biosynthesis and transcriptional regulators using both RNA-seq and quantitative PCR. The differential expression of the transcription factor genes R2R3 MYB SmuMYB113 and R3MYB SmuATV suggested their association with purple skin and foliage phenotype. Functional analysis of these genes in both tobacco and pepino showed that SmuMYB113 activates anthocyanins, while SmuATV suppresses anthocyanin accumulation. However, despite elevated expression in all tissues, SmuMYB113 does not significantly elevate flesh pigmentation, suggesting a strong repressive background in fruit flesh tissue. These results will aid understanding of the differential regulation controlling fruit quality aspects between skin and flesh in other fruiting species.

Homologs of FT, CEN and FD respond to developmental and environmental signals affecting growth and flowering in the perennial vine kiwifruit.

FLOWERING LOCUS T (FT) and CENTRORADIALIS (CEN) homologs have been implicated in regulation of growth, determinacy and flowering. The roles of kiwifruit FT and CEN were explored using a combination of expression analysis, protein interactions, response to temperature in high-chill and low-chill kiwifruit cultivars and ectopic expression in Arabidopsis and Actinidia. The expression and activity of FT was opposite from that of CEN and incorporated an interaction with a FLOWERING LOCUS D (FD)-like bZIP transcription factor. Accumulation of FT transcript was associated with plant maturity and particular stages of leaf, flower and fruit development, but could be detected irrespective of the flowering process and failed to induce precocious flowering in transgenic kiwifruit. Instead, transgenic plants demonstrated reduced growth and survival rate. Accumulation of FT transcript was detected in dormant buds and stem in response to winter chilling. In contrast, FD in buds was reduced by exposure to cold. CEN transcript accumulated in developing latent buds, but declined before the onset of dormancy and delayed flowering when ectopically expressed in kiwifruit. Our results suggest roles for FT, CEN and FD in integration of developmental and environmental cues that affect dormancy, budbreak and flowering in kiwifruit.

A MADS-box gene-induced early flowering pear (Pyrus communis L.) for accelerated pear breeding.

There have been a considerable number of studies that have successfully sped up the flowering cycle in woody perennial horticultural species. One particularly successful study in apple (Malus domestica) accelerated flowering using a silver birch (Betula pendula) APETALA1/FRUITFULL MADS-box gene BpMADS4, which yielded a good balance of vegetative growth to support subsequent flower and fruit development. In this study, BpMADS4 was constitutively expressed in European pear (Pyrus communis) to establish whether this could be used as a tool in a rapid pear breeding program. Transformed pear lines flowered within 6-18 months after grafting onto a quince (Cydonia oblonga) rootstock. Unlike the spindly habit of early flowering apples, the early flowering pear lines displayed a normal tree-like habit. Like apple, the flower appearance was normal, and the flowers were fertile, producing fruit and seed upon pollination. Seed from these transformed lines were germinated and 50% of the progeny flowered within 3 months of sowing, demonstrating a use for these in a fast breeding program.

Recurrent neo-sex chromosome evolution in kiwifruit.

Sex chromosome evolution is thought to be tightly associated with the acquisition and maintenance of sexual dimorphisms. Plant sex chromosomes have evolved independently in many lineages1,2 and can provide a powerful comparative framework to study this. We assembled and annotated genome sequences of three kiwifruit species (genus Actinidia) and uncovered recurrent sex chromosome turnovers in multiple lineages. Specifically, we observed structural evolution of the neo-Y chromosomes, which was driven via rapid bursts of transposable element insertions. Surprisingly, sexual dimorphisms were conserved in the different species studied, despite the fact that the partially sex-linked genes differ between them. Using gene editing in kiwifruit, we demonstrated that one of the two Y-chromosome-encoded sex-determining genes, Shy Girl, shows pleiotropic effects that can explain the conserved sexual dimorphisms. These plant sex chromosomes therefore maintain sexual dimorphisms through the conservation of a single gene, without a process involving interactions between separate sex-determining genes and genes for sexually dimorphic traits.

Advancing tree genomics to future proof next generation orchard production.

The challenges facing tree orchard production in the coming years will be largely driven by changes in the climate affecting the sustainability of farming practices in specific geographical regions. Identifying key traits that enable tree crops to modify their growth to varying environmental conditions and taking advantage of new crop improvement opportunities and technologies will ensure the tree crop industry remains viable and profitable into the future. In this review article we 1) outline climate and sustainability challenges relevant to horticultural tree crop industries, 2) describe key tree crop traits targeted for improvement in agroecosystem productivity and resilience to environmental change, and 3) discuss existing and emerging genomic technologies that provide opportunities for industries to future proof the next generation of orchards.

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.