The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ϵ) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene

The Phytoene synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content

Background Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation. Results The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in ‘Granny Smith’ and ‘Royal Gala’ apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms. Conclusion The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0573-7) contains supplementary material, which is available to authorized users

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

Characterisation of tomato MADS-box genes involved in flower and fruit development : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand

MADS-box genes encode transcription factors that are involved in various aspects of plant development, by regulating target genes that control morphogenesis. Over the last decade, plant MADS-box genes have been studied extensively to reveal their control of floral development, especially in the model plants Arabidopsis and Antirrhinum. Their functions are however, not restricted to the flower but are involved in various aspects of plant development (Rounsley et al., 1995; Jack, 2001). By virtue of their extensive roles in the flower, these genes are expected to function in fruit development, which is a progression from flower morphogenesis. The aim of this study was to examine the role of MADS-box genes during flower and fruit development. Two new members of the tomato MADS-box gene family, TM10 and TM29 were identified. TM29 was isolated from a young fruit cDNA library by screening with homologous MADS-box fragments and TM10 was amplified by polymerase chain reaction from fruit cDNA templates. These genes were characterised by sequence and RNA expression patterns and their functions examined using molecular genetic techniques. Sequence analyses confirmed that both genes belong to the MADS-box family. TM29 shows 68% amino acid sequence identity to Arabidopsis SEP1 MADS-box protein. TM29 expression pattern showed similarities as well as differences to SEP1 (Flanagan and Ma. 1994). TM29 is expressed in shoot, inflorescence and floral meristems unlike SEP1, which is expressed exclusively in floral meristems (Flanagan and Ma. 1994). TM29 is expressed in all the four whorls of the flower. During floral organ development, it is highly expressed at early stages of the organ primordium but decreases as the organ differentiates and matures. In the mature flower bud, TM29 is expressed in the anther and ovary pericarp. During fruit development, TM29 is expressed from anthesis ovary to fruit of 14 days post-anthesis with its transcript localised to the pericarp and placenta. TM10 showed 64% amino acid identity to Arabidopsis AGL12. across the entire sequence. This notwithstanding, TM10 expression differed from AGL12. TM10 was expressed in shoot tissues of tomato and was not detected in roots. In contrast, the AGL12 gene transcript was only present in the roots of Arabidopsis (Rounsley et al., 1995). Expression was detected in leaves, shoot growing tips, floral buds and fruit. During fruit development, TM10 is expressed in anthesis ovary and in fruits at different growth stages. The functions of TM29 and TM10 were examined by transgenic techniques and phenotypes generated were consistent with their spatial and temporal gene expression patterns. TM29 transgenic phenotypes suggested it might be involved in the control of sympodial growth, transition to flowering, proper development of floral organs. parthenocarpic fruit development and maintenance of floral meristem identity. TM10 affected apical dominance and flowering time, development of floral organs and parthenocarpic fruit development

Genetic engineering for antibacterial activity in lettuce (Lactuca sativa L.)

Lettuce crop is affected by bacterial diseases (both in the field and in storage) with severe, consequences which can be mitigated through improving disease resistance in lettuce. The numerous constraints associated with sexual crosses in lettuce render novel gene transfer via genetic manipulation a suitable method for lettuce improvement. This study was undertaken to transfer three chimaeric genes encoding antibacterial peptides into lettuce and to ascertain the effectiveness of these peptides in enhancing bacterial disease resistance. Genetic manipulation of plants requires a good regeneration ability of genotypes in tissue culture, to ensure recovery of complete transformed plants. Twenty-two lettuce genotypes belonging to several morphological groups were thus screened for their regeneration response to defined tissue culture conditions and selection of amenable genotypes for genetic manipulation. Genotypic variation was observed in callus, shoot and root production in vitro; two lettuce genotypes Bambino (a crisphead) and Cobham Green (a butterhead) with good tissue culture response were subsequently chosen for Agrobacterium-mediated transformation. Two binary vectors carrying chimaeric genes (with T4 lysozyme, magainin IT coding sequences) were obtained from Crop & Food Research, Lincoln; these vectors had pBINPLUS (a recently constructed binary plasmid) as the backbone. A third binary vector, similar in structure to the two others, with a chimaeric gene encoding Shiva-1 (a cecropin B analogue) was successfully constructed. The three vectors, pBINPLYS, pBINMGN and pBINPLUSH (encoding T4 lysozyme, magainin IT and Shiva-1 respectively) in Agrobacterium strain AGLI were used for the transformation of Bambino and Cobham Green. Two transformation treatments: a delay of 5 days (after co cultivation) before explants were transferred to kanamycin selection medium and an immediate transfer of explants to selection medium were studied for their effect on transformation frequency. Successful regenerated transformed plants of Cobham Green were obtained in this study; however, attempts to transform Bambino proved futile and no regenerated transformed shoots of Bambino were obtained. The specific reasons for the failure to transform Bambino are unknown but crisphead lettuce genotypes are often recorded as being recalcitrant to Agrobacterium-mediated transformation. The delayed selection treatment gave higher transformation frequencies in Cobham Green than the immediate selection treatment. Polymerase Chain Reaction (PCR) with specific npt IT oligonucleotide primers confirmed the presence of the selectable marker gene in all the 22 putative transgenic plants recovered. An inheritance study on the selfed progeny of transformed lines, confirmed transmission of the kanamycin resistance and pointed to a single insertion of the npt II gene in most of the transformed lettuce lines. The presence of the antibacterial genes was established in all the transformed plants, by PCR. The presence and effectiveness of the antibacterial peptides were determined by an in vitro assay and greenhouse evaluation of pathogen resistance in seedlings. Crude leaf extracts from selected transformed lines and control of untransformed Cobham Green were added to cultures of Erwinia carotovora subsp. carotovora and Xanthomonas campestris pv vitians to establish their effect on the growth of the pathogens. Growth of pathogens in cultures with crude extracts from transformed lines was inhibited while extracts from the control showed no such effect on the growth of the pathogens. In the greenhouse, selfed progeny of transformed lines and untransformed control were inoculated with the two lettuce pathogens to assess their response to disease development. Overall, delayed symptoms and reduced disease severity characterised the inoculated transformed lines, compared to the control seedlings. The potential of the antibacterial genes to effectively improve the resistance of bacterial diseases in lettuce was evident in these assays

Alternative splicing of the PECTINESTERASE gene encoding a cell wall-degrading enzyme affects postharvest softening in grape

The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes (DEGs) by comparing the transcriptomes of folic acid (FA)-treated and water-treated (CK) berries at different time points. We then analyzed the sequences to detect alternatively spliced (AS) genes associated with postharvest softening. A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group. There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively. Gene Ontology (GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes, particularly cell wall degradation processes. Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2 (VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event. The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries. Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries

Gain-of-Function Phenotypes of Many CLAVATA3/ESR Genes, Including Four New Family Members, Correlate with Tandem Variations in the Conserved CLAVATA3/ESR Domain

Secreted peptide ligands are known to play key roles in the regulation of plant growth, development, and environmental responses. However, phenotypes for surprisingly few such genes have been identified via loss-of-function mutant screens. To begin to understand the processes regulated by the CLAVATA3 (CLV3)/ESR (CLE) ligand gene family, we took a systems approach to gene identification and gain-of-function phenotype screens in transgenic plants. We identified four new CLE family members in the Arabidopsis (Arabidopsis thaliana) genome sequence and determined their relative transcript levels in various organs. Overexpression of CLV3 and the 17 CLE genes we tested resulted in premature mortality and/or developmental timing delays in transgenic Arabidopsis plants. Overexpression of 10 CLE genes and the CLV3 positive control resulted in arrest of growth from the shoot apical meristem (SAM). Overexpression of nearly all the CLE genes and CLV3 resulted in either inhibition or stimulation of root growth. CLE4 expression reversed the SAM proliferation phenotype of a clv3 mutant to one of SAM arrest. Dwarf plants resulted from overexpression of five CLE genes. Overexpression of new family members CLE42 and CLE44 resulted in distinctive shrub-like dwarf plants lacking apical dominance. Our results indicate the capacity for functional redundancy of many of the CLE ligands. Additionally, overexpression phenotypes of various CLE family members suggest roles in organ size regulation, apical dominance, and root growth. Similarities among overexpression phenotypes of many CLE genes correlate with similarities in their CLE domain sequences, suggesting that the CLE domain is responsible for interaction with cognate receptors

Table_3_Overexpression of PSY1 increases fruit skin and flesh carotenoid content and reveals associated transcription factors in apple (Malus × domestica).XLSX

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion  treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis.</p