Alteration of flower colour in <Emphasis Type="Italic">Ipomoea nil</Emphasis> through CRISPR/Cas9-mediated mutagenesis of <Emphasis Type="Italic">carotenoid cleavage dioxygenase 4</Emphasis>

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.     

Photoperiodic flower induction in <Emphasis Type="Italic">Ipomoea nil</Emphasis> is accompanied by decreasing content of gibberellins

The involvement of gibberellins (GAs) in the control of flower induction in the short-day plant Ipomoea nil has been investigated. To clarify the molecular basis of this process, we identified the full-length cDNAs of the InGA20ox3 and InGA2ox1 genes, which encode enzymes responsible for GA biosynthesis and catabolism, respectively. We studied the expression patterns of both genes and determined the tissue and cellular immunolocalisation of gibberellic acid (GA₃) in the cotyledons of 5-day-old seedlings growing under inductive and non-inductive photoperiodic conditions. In the second half of the inductive night, which is crucial for flower induction in I. nil, InGA20ox3 expression decreased, whereas InGA2ox1 mRNA accumulated, which indicates that photoperiod regulates the activity of both genes. Furthermore, these changes are correlated with GA₃ level. Thus, our results support the thesis that the proper balance between the expression of the InGA20ox3 and InGA2ox1 genes and low GA₃ content correlate with photoperiodic flower induction in I. nil.     

Identification of <Emphasis Type="Italic">r</Emphasis> mutations conferring white flowers in the Japanese morning glory (<Emphasis Type="Italic">Ipomoea nil</Emphasis>)

The wild-type Japanese morning glory [Ipomoea nil (L.) Roth.] exhibits blue flowers with red stems, and spontaneous r mutants display white flowers with green stems. We have identified two r mutations, r1-1 and r1-2, that are caused by insertions of Tpn1-related DNA transposable elements, Tpn3 (5.6 kb) and Tpn6 (4.7 kb), respectively, into a unique intron of the CHS-D gene, which is responsible for flower and stem pigmentation. Both Tpn3 and Tpn6, which belong to the En/Spm or CACTA superfamily, are nonautonomous elements lacking transposase genes but containing unrelated cellular DNA segments including exons and introns. Interestingly, r1-2 contains an additional 4-bp insertion at the Tpn3 integration site in r1-1, presumably a footprint caused by the excision of Tpn3. The results strengthen the previous notion that Tpn1 and its relatives are major spontaneous mutagens for generating various floriculturally important traits in I. nil. Since I. nil has an extensive history of genetic studies, molecular identification of classical spontaneous mutations would also facilitate reinterpretation of the abundant classical genetic data available.     

Inhibition of root respiration induces leaf senescence in <Emphasis Type="Italic">Alhagi sparsifolia</Emphasis>

Leaf senescence can be induced by numerous factors. In order to explore the relationship between root respiration and leaf senescence, we utilized different types of phloem girdling to control the root respiration of Alhagi sparsifolia and its physiological response. Our results showed that both girdling and inhibition of root respiration led to a decline of stomatal conductance, photosynthesis, transpiration rate, chlorophyll (Chl) a, Chl b, carotenoid (Car) content, Chl a/b, Chl/Car, water potential, and Chl a fluorescence, as well as to an increase of abscisic acid (ABA), proline, and malondialdehyde content in leaves and to upregulation of senescence-associated gene expression. Our present work implied that both inhibition of root respiration and girdling can induce leaf senescence. In comparison with phloem girdling, the leaf senescence caused by inhibition of root respiration was less significant. The reason for girdling-induced senescence was ABA and carbohydrate accumulation. Senescence induced by inhibition of root respiration occurred due to leaf water stress resulting from inhibition of water absorption.     

Expression of <Emphasis Type="Italic">IPT</Emphasis> in Asakura-sanshoo (<Emphasis Type="Italic">Zanthoxylum piperitum</Emphasis> (L.) DC. f. <Emphasis Type="Italic">inerme</Emphasis> Makino) Alters Tree Architecture,Delays Leaf Senescence,and Changes Leaf Essential Oil Composition

The IPT gene encodes isopentenyl pyrophosphate transferase, a key enzyme in cytokinin biosynthesis. We introduced IPT under the control of the CaMV35S promoter into Asakura-sanshoo (Zanthoxylum piperitum (L.) DC. f. inerme Makino) via stable Agrobacterium tumefaciens-mediated transformation. Three of 3-year-old transgenic Asakura-sanshoo lines Y5, Y16, and Y17 were used to evaluate the effects of IPT expression on the morphological characteristics, leaf senescence, and essential oil composition. Introduced IPT into Asakura-sanshoo stimulated an increase in cytokinin content and a decrease in auxin level. The increase in the cytokinin/auxin ratio affected the tree architecture in 3-year-old transgenic lines. The phenotypes of transgenic lines included reduced stem elongation, decreased leaf surface area, increased branching, and delayed leaf senescence. The expression of IPT in Asakura-sanshoo also affected the leaf essential oil composition. The amount of oxygenated sesquiterpenoid compounds in Y5 and Y16 was 21.1 and 15.8 % higher, respectively, than that in wild type (WT). The amount of aromatic compounds in Y5 and Y16 was 2.9 and 24.6 % lower, respectively, than that in WT. These results show that ipt expression in Asakura-sanshoo conferred desirable traits, including a dwarf growth habit, delayed senescence, and increased concentrations of some sesquiterpenoid compounds.     

The stay-green phenotype of wheat mutant <Emphasis Type="Italic">tasg1</Emphasis> is associated with altered cytokinin metabolism

Key message

By measuring the cytokinin content directly and testing the sensitivity to the cytokinin inhibitor lovastatin, we demonstrated that tasg1 cytokinin metabolism is different from wild-type.

Abstract

Our previous studies have indicated that compared with wild-type (WT) plants, a wheat stay-green mutant tasg1 exhibited delayed senescence. In this study, we found that the root development of tasg1 occurred later than that of WT. The number of lateral roots was fewer, but the lateral root length was longer in tasg1 than in WT, which resulted in a lower root to shoot ratio in tasg1 than WT. The levels of cytokinin (CK), CK activity, and expression of CK metabolic genes were measured. We found that the total CK content in the root tips and leaf of tasg1 was greater than in WT. The accumulation of mRNA of the CK synthetic gene (TaIPT) in tasg1 was higher than in WT at 9 and 11 days during seedling growth, but the expression of CK oxidase gene (TaCKX) was significantly lower in tasg1. Furthermore, the CK inhibitor lovastatin was used to inhibit CK activity. When treated with lovastatin, both the chlorophyll content and thylakoid membrane protein stability were significantly lower in tasg1 than WT, consistent with the inhibited expression of senescence-associated genes (TaSAGs) in tasg1. Lovastatin treatment also inhibited the antioxidative capability of wheat seedlings, and tasg1 was more sensitive to lovastatin than WT, as indicated by the MDA content, protein carbonylation, and antioxidant enzyme activity. The decreased antioxidative capability after lovastatin treatment may be related to the down-regulation of some antioxidase genes. These results suggest that the CK metabolism was altered in tasg1, which may play an important role in its ability to delay senescence.

     

The rice <Emphasis Type="Italic">OsSAG12-2</Emphasis> gene codes for a functional protease that negatively regulates stress-induced cell death

Senescence is the final stage of plant development. Although expression of most of the genes is suppressed during senescence, a set of genes referred as senescence-associated genes (SAGs) is induced. Arabidopsis thaliana SAG12 (AtSAG12) is one such gene that has been mostly studied for its strict association with senescence. AtSAG12 encodes a papain-like cysteine protease, expressed predominantly in senescence-associated vacuoles. Rice genome contains multiple AtSAG12 homologues (OsSAGs). OsSAG12-1, the closest structural homologue of AtSAG12, is a negative regulator of developmental and stress-induced cell death. Proteolytic activity has not been established for any SAG12 homologues in vitro. Here, we report that OsSAG12-2, the second structural homologue of AtSAG12 from rice, codes for a functional proteolytic enzyme. The recombinant OsSAG12-2 protein produced in Escherichia coli undergoes autolysis to generate a functional protease. The matured OsSAG12-2 protein shows 27% trypsin-equivalent proteolytic activity on azocasein substrate. Dark-induced senescence activates OsSAG12-2 expression. Down-regulation of OsSAG12-2 in the transgenic artificial miRNA lines results in enhanced salt- and UV-induced cell death, even though it does not affect cell viability in the stress-free condition. Our results show that OsSAG12-2 codes for a functional protease that negatively regulates stress-induced cell death in rice.     

Pollination-induced floral senescence in orchids: Status of oxidative stress

The orchid flowers may stay fresh in unpollinated state from few weeks to months but show rapid senescence upon pollination. Metabolic changes related to this phenomenon are less well understood in orchid flowers. Presently, two orchid species, Aerides multiflora Roxb. and Rhynchostylis retusa (L.) Bl., varying in their floral life span were evaluated for their postpollination-induced responses, involving the oxidative stress. The unpollinated flowers of A. multiflora stayed fresh for 17 days and attained senescence in 5 days after pollination (DAP), while those of R. retusa. remained fresh for 24 days and showed senescence in 7 DAP. After pollination, wilting began in 2 to 3 days in A. multiflora and 3 to 4 days in R. retusa. There was a higher electrolyte leakage accompanied by a concomitant increase in the levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), indicators of oxidative damage in all the organs after pollination while ascorbic acid decreased significantly. The flowers of A. multiflora showed a greater electrolyte leakage, MDA and H₂O₂ contents as compared to those of R. retusa. Ascorbic acid content, on the other hand, was lower in A. multiflora than in R. retusa, suggesting a higher oxidative damage to the floral organs in the former species. An application of triiodobenzoic acid ( an auxin inhibitor; 0.25 mM) and silver nitrate (ethylene inhibitor; 0.25 mM) to pollinated flowers partially prevented the oxidative damage and consequently the senescence, suggesting the involvement of these hormones. AgNO₃ was more effective in delaying senescence.     

Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous <Emphasis Type="Italic">Rhodococcus</Emphasis> strains

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

A scientific note on the evidence for the production and use of wax by a captive female <Emphasis Type="Italic">Bombus</Emphasis><Emphasis Type="Italic">sylvestris</Emphasis>

The presence of wax glands in the socially parasitic subgenus Bombus (Psithyrus) was established only recently (Sramkova and Ayasse, 2008), but the uses for the produced wax are unknown. In this investigation nest-searching Bombus sylvestris females were collected and kept in darkness, in conditions of controlled temperature and relative humidity, using rearing conditions for non-parasitic bumblebees. The results show that in Bombus sylvestris not only wax glands are present but they also can produce wax which can be used as a building material.     

Daily activity rhythm of desert omni-seasonal Tenebrionid beetles, <Emphasis Type="Italic">Trigonoscelis gigas</Emphasis> and <Emphasis Type="Italic">T. sublaevicollis</Emphasis> (Coleoptera,Tenebrionidae) in constant darkness and under alternating 1-hour pulses of light and darkness

In the Karakum Desert (Turkmenistan) the beetles Trigonoscelis gigas are only active in the morning and evening while T. sublaevicollis are strictly nocturnal, regardless of the season and weather. The daily activity rhythm of T. gigas and T. sublaevicollis was studied in the laboratory according to the following pattern: 5 days under a light-darkness cycle of 15: 9 h (LD 15: 9), then 10 days in constant darkness (DD), and then 10 more days under alternating 1-h pulses of light and darkness (LD 1: 1). The temperature was 25°C in all the modes. At LD 15: 9, beetles of both species maintained a 24-h period and a natural pattern of the activity rhythm. In DD, the circadian rhythm ran with a period of 23.5 ± 0.3 h (n = 40) in T. gigas and 23.6 ± 0.4 h (n = 40) in T. sublaevicollis. At DD, the morning and evening activity peaks of T. gigas merged to form a rhythm with only one peak. Under LD 1: 1, both T. gigas and T. sublaevicollis recovered a 24-h period of the rhythm, while the rhythm of T. gigas regained the two-peak structure. Our research confirmed the assumption of Tshernyshev (1980) about the 24-h period of the free-running endogenous rhythm and the distorting effect of constant conditions on this rhythm.     

Effects of endogenous salicylic acid synthesized through PAL and ICS pathway on baicalin and baicalein accumulation in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

The aim of this study was to investigate the effect of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS) on free salicylic acid (FSA) or total salicylic acid (TSA) content, and the effect of endogenous SA on baicalin and baicalein accumulation in Scutellaria baicalensis Georgi, respectively. We amplified partial sequences of PAL and ICS genes in Scutellaria baicalensis Georgi and silenced the two genes with virus-induced gene silence (VIGS) technique, respectively. The influence of gene silence on FSA, TSA, baicalin, and baicalein accumulation in Scutellaria baicalensis Georgi were analyzed, and these parameters were also investigated under high temperature. Results indicated that PAL silence significantly affected the FSA, ICS affected TSA content. FSA significantly affected the baicalin, rather than baicalein content. Our results along with previous studies indicated PAL and ICS were different in the regulation of FSA or TSA synthesis, and FSA and TSA were different in the regulation of baicalin and baicalein synthesis in Scutellaria baicalensis Georgi.     

Effect of exogenous abscisic acid on cold acclimation in two Magnolia species

In northern China, freezing injury is observed frequently in the rare species Magnolia wufengensis but not in the more common species Magnolia denudata. To investigate the role of the phytohormone abscisic acid (ABA) on frost tolerance in these two species, exogenous ABA was applied to the seedlings and then physiological and biochemical responses were measured during cold acclimation. Shoot growth cessation was stimulated by ABA in M. wufengensis but not in M. denudata. Abscisic acid inhibited shoot growth in M. wufengensis but not in M. denudata. Treatment with ABA stimulated leaf senescence in both species, and this effect was greater in M. denudata. For both species, ABA-treated plants exhibited bud dormancy sooner and had an increased tolerance to freezing, decreased water content and increased accumulation of proline, glucose, and fructose in shoots. These effects were generally greater for M. denudata. Freezing tolerance was significantly correlated with content of water, proline, glucose, and fructose for both species, but freezing tolerance was significantly correlated with raffinose content only in M. wufengensis. We conclude that exogenous ABA could increase cold acclimation and improve cold hardiness of both Magnolia species, although M. denudata was more responsive to ABA than M. wufengensis, which might result from a greater dehydration and accumulation of proline and certain soluble sugars.     

The composition of fatty acids in the endosperm and embryo lipids of <Emphasis Type="Italic">Pinus sibirica</Emphasis> and <Emphasis Type="Italic">P. sylvestris</Emphasis> seeds

The fatty acid (FA) composition of storage lipids in the seed endosperms and embryos of two pine species, Pinus sibirica and P. sylvestris, and possible biosynthetic pathways of these acids were studied by the GLC method. Linoleic acid predominated in the embryo and endosperm lipids of both P. sibirica (43.5 and 42.6%) and P. sylvestris (44.8 and 46.8%); this was evidently determined by a high expression of the gene encoding stearoyl-Δ9 acyl-lipid desaturase and the fad2 gene encoding microsomal ω6 acyl-lipid desaturase. P. sibirica lipids of the embryo and endosperm contained more oleic acid (22.0 and 24.0%, respectively) than corresponding P. sylvestris lipids (18.7 and 14%). Storage lipids of conifer seeds contain Δ5-unsaturated FAs: taxoleic (18:2Δ5,9), ephedrenic (18:2Δ5,11), pinoleenic (18:3Δ5,9,12), skiadonic (18:3Δ5,11, 14), and coniferonic (18:4Δ5,9,12,15). In the endosperm and embryos of P. sylvestris, the content of pinolenic acid was higher (22.1 and 19.6%) than in P. sibirica seeds (19.1 and 18.6%).