Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap

<p>Abstract</p> <p>Background</p> <p>Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks.</p> <p>Results</p> <p>Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (<it>Malus </it>x <it>domestica </it>'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs.</p> <p>Conclusions</p> <p>This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants.</p

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

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

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164\ua0Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models

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 l 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

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

<div><p>Trehalose metabolism and its intermediate trehalose-6-phosphate (T6P) are implicated in sensing and signalling sucrose availability. Four class I <i>TREHALOSE-6-PHOSPHATE SYNTHASE</i> (<i>TPS1</i>) 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 <i>TPS1</i>, designated <i>TPS1</i>.<i>1a</i> was the most highly abundant <i>TPS1</i> transcript in all examined kiwifruit tissues. An additional exon giving rise to a small N-terminal extension was found for two of the <i>TPS1</i> transcripts, designated <i>TPS1</i>.<i>2a</i> and <i>TPS1</i>.<i>2b</i>. Homology in sequence and gene structure with <i>TPS1</i> 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 <i>TPS1</i>.<i>2</i> transcripts was sufficient to substitute for the lack of functional TPS1 in the yeast <i>tps1Δ tps2Δ</i> mutant, but only weak complementation was detected in the yeast <i>tps1Δ</i> mutant, and no or very weak complementation was obtained with the <i>TPS1</i>.<i>1a</i> construct. Transgenic Arabidopsis lines expressing kiwifruit <i>TPS1</i>.<i>2</i> under the control of 35S promoter exhibited growth and morphological defects. We investigated the responses of plants to elevated kiwifruit <i>TPS1</i> activity at the transcriptional level, using transient expression of <i>TPS1</i>.<i>2a</i> in <i>Nicotiana benthamiana</i> leaves, followed by RNA-seq. Differentially expressed genes were identified as candidates for future functional analyses.</p></div

Phenotypes of transgenic Arabidopsis constitutively expressing <i>TPS1</i> genes.

<p>A. Early growth on ½ MS medium supplemented with sucrose and kanamycin. B. Plants grown in soil.</p

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

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