Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

<p>Abstract</p> <p>Background</p> <p>Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding.</p> <p>Results</p> <p>The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of <it>Solanum tuberosum </it>(potato) and petals of <it>Antirrhinum majus</it>. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from <it>Amanita muscaria</it>) or a plant DOD (from <it>Portulaca grandiflora</it>), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed <it>Arabidopsis thaliana </it>(arabidopsis) lines containing <it>35S: AmDOD </it>produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production.</p> <p>Conclusions</p> <p>The fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to <it>cyclo</it>-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed <it>35S: AmDOD </it>arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.</p

The Cytokinin Complex Associated With Rhodococcus fascians: Which Compounds Are Critical for Virulence?

Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms

Revival of the magnetar PSR J1622-4950: observations with MeerKAT, Parkes, XMM-Newton, Swift, Chandra, and NuSTAR

New radio (MeerKAT and Parkes) and X-ray (XMM-Newton, Swift, Chandra, and NuSTAR) observations of PSR J1622-4950 indicate that the magnetar, in a quiescent state since at least early 2015, reactivated between 2017 March 19 and April 5. The radio flux density, while variable, is approximately 100x larger than during its dormant state. The X-ray flux one month after reactivation was at least 800x larger than during quiescence, and has been decaying exponentially on a 111+/-19 day timescale. This high-flux state, together with a radio-derived rotational ephemeris, enabled for the first time the detection of X-ray pulsations for this magnetar. At 5%, the 0.3-6 keV pulsed fraction is comparable to the smallest observed for magnetars. The overall pulsar geometry inferred from polarized radio emission appears to be broadly consistent with that determined 6-8 years earlier. However, rotating vector model fits suggest that we are now seeing radio emission from a different location in the magnetosphere than previously. This indicates a novel way in which radio emission from magnetars can differ from that of ordinary pulsars. The torque on the neutron star is varying rapidly and unsteadily, as is common for magnetars following outburst, having changed by a factor of 7 within six months of reactivation.Comment: Published in ApJ (2018 April 5); 13 pages, 4 figure

A study on the role of cytokinins in the development of starch accumulating structures.

Cytokinin-like activity and soluble sugar and starch levels were monitored during wheat grain (Triticum aestivum L.) and potato tuber (Solanum spp) development. Cytokinin-like activity was resolved on Sephadex LH-20 eluted with 35 or 20% ethanol and estimated in kinetin in equivalents from the soybean callus bioassay. The cytokinin-like activity increased in tubers larger than 5 - 7.5 mm diameter and reached a maximum in tubers 15 - 20 mm diameter. The amount per tuber was greatest in the largest size category analysed (40-50 mm diameter). The amount of sugar and starch per tuber also increased after tuber formation. There was a positive correlation between the highest concentration of cytokinin-like activity and the reported period of intense cell division, The cytokinin-like activity per pistil and per unit weight increased between ear emergence and pollination. On a per grain basis the activity increased to a high level 14 days after ear emergence (four days post-anthesis) but subsequently decreased to an undetectable level by 21 days. On a per unit weight basis the concentration was high but fluctuated between 10 and 14 days after ear emergence before decreasing. The most polar components of the activity were the O-glucosides of zeatin and zeatin riboside. These showed a rapid increase between ear emergence and anthesis but subsequently decreased, whereas zeatin increased most rapidly following pollination and reached a maximum four days post-anthesis. Zeatin riboside remained at a relatively low level at all stages of development. The highest level of cytokinin-like activity correlated with the reported onset of normal cell divisions in the endosperm. The amount of activity remained low as sugar and starch levels in the grain increased. Both zeatin and zeatin riboside were positively identified by GCMS (MIM) in extract of wheat grains

Effects of three plant growth regulators on growth, morphology, water relations, and frost resistance in lemonwood (Pittosporum eugenioides A.Cunn)

Pittosporum eugenioides is a native ornamental species with a wide distribution throughout New Zealand. The effects of the application of two plant hormones (gibberellic acid and abscisic acid) and a gibberellin biosynthesis inhibitor (paclobutrazol) on the growth and development, frost resistance, and water loss on two-year old seedlings of P. eugenioides were studied.Gibberellic acid increased plant growth, stem diameter, and shoot succulence, while also inducing leaf hyponasty and vegetative bud break of apices. Abscisic acid induced growth suppression while being effective in promoting leaf epinasty and inhibiting bud break. Paclobutrazol induced growth suppression, promoted epinasty, decreased shoot succulence, inhibited bud break in apices, and increased foliar frost resistance. The effects of paclobutrazol were also overcome by the addition of gibberellic acid. The results are compared with those gained from species not native to New Zealand, and indicate the potential for the application of plant growth regulators to a New Zealand species of ornamental value

Cytokinins and Expression of SWEET, SUT, CWINV and AAP Genes Increase as Pea Seeds Germinate

Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT), amino acid permease (AAP), Sugar Will Eventually be Exported Transporter (SWEET), cell wall invertase (CWINV), cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration

Differential Gene Expression in the Meristem and during Early Fruit Growth of Pisum sativum L. Identifies Potential Targets for Breeding

For successful molecular breeding it is important to identify targets to the gene family level, and in the specific species of interest, in this case Pisum sativum L. The cytokinins have been identified as a key breeding target due to their influence on plant architecture, and on seed size and sink activity. We focused on the cytokinin biosynthetic gene family (the IPTs) and the gene family key to the destruction of cytokinins (the CKXs), as well as other gene families potentially affected by changing cytokinin levels. These included key meristem genes (WUS and BAM1) and the transporter gene families, sucrose transporters (SUTs) and amino acid permeases (AAPs). We used reverse transcription quantitative PCR (RT-qPCR) to monitor gene expression in the vegetative meristem and in pre- and post-fertilisation young pea fruits. PsWUS expression was specific to the shoot apical meristem while PsBAM1 was highly expressed in the shoot apical meristem (SAM) but was also expressed at a low level in the young fruit. Differential expression was shown between genes and within gene families for IPT, CKX, SUT, and AAP. PsCKX7 showed strong gene family member-specific expression in the SAM, and was also expressed in young pea fruits. We suggest that PsCKX7 is a potential target for downregulation via molecular breeding or gene editing

Plant Growth Regulators INCYDE and TD-K Underperform in Cereal Field Trials

Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels

Plant Growth Regulators INCYDE and TD-K Underperform in Cereal Field Trials

Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels

Identification of flowering-time genes in mast flowering plants using De Novo transcriptomic analysis.

Mast flowering is synchronised highly variable flowering by a population of perennial plants over a wide geographical area. High seeding years are seen as a threat to native and endangered species due to high predator density caused by the abundance of seed. An understanding of the molecular pathways that influence masting behaviour in plants could provide better prediction of a forthcoming masting season and enable conservation strategies to be deployed. The goal of this study was to identify candidate flowering genes that might be involved in regulating mast flowering. To achieve this, high-throughput large-scale RNA-sequencing was performed on two masting plant species, Celmisia lyallii (Asteraceae), and Chionochloa pallens (Poaceae) to develop a reference transcriptome for functional and molecular analysis. An average total of 33 million 150 base-paired reads, for both species, were assembled using the Trinity pipeline, resulting in 151,803 and 348,649 transcripts respectively for C. lyallii and C. pallens. For both species, about 56% of the unigenes were annotated with gene descriptions to known proteins followed by Gene Ontology analysis, categorising them on the basis of putative biological processes, molecular function, and cellular localization. A total of 543 transcripts from C. lyallii and 470 transcripts from C. pallens were also mapped to unique flowering-time proteins identified in Arabidopsis thaliana, suggesting the conservation of the flowering network in these wild alpine plants growing in natural field conditions. Expression analysis of several selected homologous flowering-pathway genes showed seasonal and photoperiodic variations. These genes can further be analysed to understand why seasonal cues, such as the increasing photoperiod in spring, that triggers the annual flowering of most plants, are insufficient to always trigger flowering in masting plants and to uncover the molecular basis of how additional cues (such as temperature during the previous growing seasons) then determines flowering in mast years