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.

Re-programming of Pseudomonas syringae pv. actinidiae gene expression during early stages of infection of kiwifruit.

BACKGROUND: Pseudomonas syringae is a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these, P. syringae pv. actinidiae (Psa) initiated a worldwide pandemic in 2008 on cultivars of Actinidia chinensis var. chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis of Psa during the early stages of kiwifruit infection. RESULTS: Gene expression in Psa was investigated during the first five days after infection of kiwifruit plantlets, using RNA-seq. Principal component and heatmap analyses showed distinct phases of gene expression during the time course of infection. The first phase was an immediate transient peak of induction around three hours post inoculation (HPI) that included genes that code for a Type VI Secretion System and nutrient acquisition (particularly phosphate). This was followed by a significant commitment, between 3 and 24 HPI, to the induction of genes encoding the Type III Secretion System (T3SS) and Type III Secreted Effectors (T3SE). Expression of these genes collectively accounted for 6.3% of the bacterial transcriptome at this stage. There was considerable variation in the expression levels of individual T3SEs but all followed the same temporal expression pattern, with the exception of hopAS1, which peaked later in expression at 48 HPI. As infection progressed over the time course of five days, there was an increase in the expression of genes with roles in sugar, amino acid and sulfur transport and the production of alginate and colanic acid. These are both polymers that are major constituents of extracellular polysaccharide substances (EPS) and are involved in biofilm production. Reverse transcription-quantitative PCR (RT-qPCR) on an independent infection time course experiment showed that the expression profile of selected bacterial genes at each infection phase correlated well with the RNA-seq data. CONCLUSIONS: The results from this study indicate that there is a complex remodeling of the transcriptome during the early stages of infection, with at least three distinct phases of coordinated gene expression. These include genes induced during the immediate contact with the host, those involved in the initiation of infection, and finally those responsible for nutrient acquisition.

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.

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.

Two Subclasses of Differentially Expressed TPS1 Genes and Biochemically Active TPS1 Proteins May Contribute to Sugar Signalling in Kiwifruit Actinidia chinensis.

Trehalose metabolism and its intermediate trehalose-6-phosphate (T6P) are implicated in sensing and signalling sucrose availability. Four class I TREHALOSE-6-PHOSPHATE SYNTHASE (TPS1) genes were identified in kiwifruit, three of which have both the TPS and trehalose-6-phosphate phosphatase (TPP) domain, while the fourth gene gives rise to a truncated transcript. The transcript with highest sequence homology to Arabidopsis TPS1, designated TPS1.1a was the most highly abundant TPS1 transcript in all examined kiwifruit tissues. An additional exon giving rise to a small N-terminal extension was found for two of the TPS1 transcripts, designated TPS1.2a and TPS1.2b. Homology in sequence and gene structure with TPS1 genes from Solanaceae suggests they belong to a separate, asterid-specific class I TPS subclade. Expression of full-length and potential splice variants of these two kiwifruit TPS1.2 transcripts was sufficient to substitute for the lack of functional TPS1 in the yeast tps1Δ tps2Δ mutant, but only weak complementation was detected in the yeast tps1Δ mutant, and no or very weak complementation was obtained with the TPS1.1a construct. Transgenic Arabidopsis lines expressing kiwifruit TPS1.2 under the control of 35S promoter exhibited growth and morphological defects. We investigated the responses of plants to elevated kiwifruit TPS1 activity at the transcriptional level, using transient expression of TPS1.2a in Nicotiana benthamiana leaves, followed by RNA-seq. Differentially expressed genes were identified as candidates for future functional analyses.