Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.

Plant hormone profiling of scion and rootstock incision sites and intra- and inter-family graft junctions in Nicotiana benthamiana.

Many previous studies have suggested that various plant hormones play essential roles in the grafting process. In this study, to understand the plant hormones that accumulate in the graft junctions, whether these are supplied from the scion or rootstock, and how these hormones play a role in the grafting process, we performed a hormonome analysis that accumulated in the incision site of the upper plants from the incision as "ungrafted scion" and lower plants from the incision as "ungrafted rootstock" in Nicotiana benthamiana. The results revealed that indole-3-acetic acid (IAA) and gibberellic acid (GA), which regulate cell division; abscisic acid (ABA) and jasmonic acid (JA), which regulate xylem formation; cytokinin (CK), which regulates callus formation, show different accumulation patterns in the incision sites of the ungrafted scion and rootstock. In addition, to try discussing the differences in the degree and speed of each event during the grafting process between intra- and inter-family grafting by determining the concentration and accumulation timing of plant hormones in the graft junctions, we performed hormonome analysis of graft junctions of intra-family grafted plants with N. benthamiana as scion and Solanum lycopersicum as rootstock (Nb/Sl) and inter-family grafted plants with N. benthamiana as scion and Arabidopsis thaliana as rootstock (Nb/At), using the ability of Nicotiana species to graft with many plant species. The results revealed that ABA and CK showed different accumulation timings; IAA, JA, and salicylic acid (SA) showed similar accumulation timings, while different accumulated concentrations in the graft junctions of Nb/Sl and Nb/At. This information is important for understanding the molecular mechanisms of plant hormones in the grafting process and the differences in molecular mechanisms between intra- and inter-family grafting.

Differences in xylem development between Dutch and Japanese tomato (Solanum lycopersicum) correlate with cytokinin levels in hypocotyls.

BACKGROUND AND AIMS: Dutch tomato cultivars tend to have a greater yield than Japanese cultivars even if they are grown under the same conditions. Factors contributing to the increased yield of the Dutch cultivars were a greater light use efficiency and greater leaf photosynthetic rate. On the other hand, the relationship between tomato yields and anatomical traits is still unclear. The aim of this study is to identify the anatomical traits related to the difference in yield between Dutch and Japanese cultivars. METHODS: Anatomical properties were compared during different growth stages of Dutch and Japanese tomatoes. Hormone profiles and related gene expression in hypocotyls of Dutch and Japanese cultivars were compared in the hypocotyls of 3- and 4-week-old plants. KEY RESULTS: Dutch cultivars have a more developed secondary xylem than Japanese cultivars, which would allow for greater transport of water, mineral nutrients and phytohormones to the shoots. The areas and ratios of the xylem in the hypocotyls of 3- to 6-week-old plants were larger in the Dutch cultivars. In reciprocal grafts of the Japanese and Dutch cultivars, xylem development at the scion and rootstock depended on the scion cultivar, suggesting that some factors in the scion are responsible for the difference in xylem development. The cytokinin content, especially the level of N6-(Δ 2-isopentenyl) adenine (iP)-type cytokinin, was higher in the Dutch cultivars. This result was supported by the greater expression of Sl-IPT3 (a cytokinin biosynthesis gene) and Sl-RR16/17 (a cytokinin-responsive gene) in the Dutch cultivars. CONCLUSIONS: These results suggest that iP-type cytokinins, which are locally synthesized in the hypocotyl, contribute to xylem development. The greater xylem development in Dutch cultivars might contribute to the high yield of the tomato.

Columnar growth phenotype in apple results from gibberellin deficiency by ectopic expression of a dioxygenase gene.

The apple cultivar McIntosh Wijcik, which is a mutant of 'McIntosh', exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, 'McIntosh Wijcik' contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2.

Carbon availability is a major regulatory factor in plant growth and development. Cytokinins, plant hormones that play important roles in various aspects of growth and development, have been implicated in the carbon-dependent regulation of plant growth; however, the details of their involvement remain to be elucidated. Here, we report that sugar-induced cytokinin biosynthesis plays a role in growth enhancement under elevated CO2 in Arabidopsis thaliana. Growing Arabidopsis seedlings under elevated CO2 resulted in an accumulation of cytokinin precursors that preceded growth enhancement. In roots, elevated CO2 induced two genes involved in de novo cytokinin biosynthesis: an adenosine phosphate-isopentenyltransferase gene, AtIPT3, and a cytochrome P450 monooxygenase gene, CYP735A2. The expression of these genes was inhibited by a photosynthesis inhibitor, DCMU, under elevated CO2, and was enhanced by sugar supplements, indicating that photosynthetically generated sugars are responsible for the induction. Consistently, cytokinin precursor accumulation was enhanced by sugar supplements. Cytokinin biosynthetic mutants were impaired in growth enhancement under elevated CO2, demonstrating the involvement of de novo cytokinin biosynthesis for a robust growth response. We propose that plants employ a system to regulate growth in response to elevated CO2 in which photosynthetically generated sugars induce de novo cytokinin biosynthesis for growth regulation.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding.

Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice-specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice-specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Effects of overproduced ethylene on the contents of other phytohormones and expression of their key biosynthetic genes.

Ethylene is involved in regulation of various aspects of plant growth and development. Physiological and genetic analyses have indicated the existence of crosstalk between ethylene and other phytohormones, including auxin, cytokinin (CK), abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR) and strigolactone (SL) in regulation of different developmental processes. However, the effects of ethylene on the biosynthesis and contents of these hormones are not fully understood. Here, we investigated how overproduction of ethylene may affect the contents of other plant hormones using the ethylene-overproducing mutant ethylene-overproducer 1 (eto1-1). The contents of various hormones and transcript levels of the associated biosynthetic genes in the 10-day-old Arabidopsis eto1-1 mutant and wild-type (WT) plants were determined and compared. Higher levels of CK and ABA, while lower levels of auxin, SA and GA were observed in eto1-1 plants in comparison with WT, which was supported by the up- or down-regulation of their biosynthetic genes. Although we could not quantify the BR and SL contents in Arabidopsis, we observed that the transcript levels of the potential rate-limiting BR and SL biosynthetic genes were increased in the eto1-1 versus WT plants, suggesting that BR and SL levels might be enhanced by ethylene overproduction. JA level was not affected by overproduction of ethylene, which might be explained by unaltered expression level of the proposed rate-limiting JA biosynthetic gene allene oxide synthase. Taken together, our results suggest that ET affects the levels of auxin, CK, ABA, SA and GA, and potentially BR and SL, by influencing the expression of genes involved in the rate-limiting steps of their biosynthesis.

Systemic transport of trans-zeatin and its precursor have differing roles in Arabidopsis shoots.

Organ-to-organ signal transmission is essential for higher organisms to ensure coordinated biological reactions during metabolism and morphogenesis. Similar to organs in animals, plant organs communicate by various signalling molecules. Among them, cytokinins, a class of phytohormones, play a key role as root-to-shoot long-distance signals, regulating various growth and developmental processes in shoots1,2. Previous studies have proposed that trans-zeatin-riboside, a type of cytokinin precursor, is a major long-distance signalling form in xylem vessels and its action depends on metabolic conversion via the LONELY GUY enzyme in proximity to the site of action3-5. Here we report an additional long-distance signalling form of cytokinin: trans-zeatin, an active form. Grafting between various cytokinin biosynthetic and transportation mutants revealed that root-to-shoot translocation of trans-zeatin, a minor component of xylem cytokinin, controls leaf size but not meristem activity-related traits, whereas that of trans-zeatin riboside is sufficient for regulating both traits. Considering the ratio of trans-zeatin to trans-zeatin-riboside in xylem and their delivery rate change in response to environmental conditions, this dual long-distance cytokinin signalling system allows plants to fine-tune the manner of shoot growth to adapt to fluctuating environments.

Lack of Cytosolic Glutamine Synthetase1;2 Activity Reduces Nitrogen-Dependent Biosynthesis of Cytokinin Required for Axillary Bud Outgrowth in Rice Seedlings.

A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in phloem companion cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.

[Anesthetic Management of Cesarean Section in a Pregnant Woman with Advanced Tongue Cancer].

It is very difficult to decide the best time to deliver the baby for a pregnant woman with advanced cancer. We experienced the perioperative and perinatal management of a 39-year-old pregnant woman with advanced tongue cancer. The cancer had already metastasized to the lung and lymph nodes. Furthermore a recurrent thumb-sized tumor was found in her mouth. She had firmly desired to discontinue all anticancer treatment for protecting the fetus. On the other hand, her family could not accept her determination yet. Therefore the medical team was organized with doctors and co-medicals from multiple departments such as gynecology, pediatrics, radiology, oncology, midwife, psychotherapy and anesthesiology. After several conferences including herself and family, finally cesarean section was scheduled for the 30th gestational week. Prepared for unexpected emergency delivery, airway stenosis was ruled out by fiberoptic laryngoscopy and the consent for emergency tracheostomy was obtained. The operation was performed successfully under spinal anesthesia without any severe troubles. Medical care as a team from early phase enabled elaborate observation and preparation through the perioperative and perinatal period. Furthermore, it was efficient to provide satisfaction to the patient and her family as well.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

[Hypotonic Versus Isotonic Electrolyte Solution for Perioperative Fluid Therapy in Infants].

BACKGROUND: This study was designed to evaluate the effects of perioperative administration of an isotonic electrolyte solution with 1% glucose (IT) on blood sodium (Na+) and blood glucose (BG) concentrations in pediatric patients < 1-year-old undergoing plastic surgery in comparison with a conventional hypotonic electrolyte solution with 2.6% glucose (HT). METHODS: Fifty Patients were randomly allocated to HT group and IT group. Na+ and BG were measured at induction of anesthesia (Tind), the end of surgery (Tend), and 4 hours after surgery (T4h). RESULTS: Patient characteristics were similar for the 2 groups. In the HT group, Na+ at Tend insignificantly dropped compared with that at Tind, whereas in the IT group Na+ was significantly elevated. Na+ at T4h significantly increased compared with that at Tend in each group. No cases developed new dysnatremia or dysglycemia in IT group. The incidence of hyponatremia at Tend was significantly lower in the IT group. A positive correlation between intraoperative Na+ concentration changes and the infusion duration was observed in the IT group. CONCLUSIONS: Isotonic solution with 1% glucose is suggested to be safe in infants during and after surgery.

Ethylene suppresses tomato (Solanum lycopersicum) fruit set through modification of gibberellin metabolism.

Fruit set in angiosperms marks the transition from flowering to fruit production and a commitment to seed dispersal. Studies with Solanum lycopersicum (tomato) fruit have shown that pollination and subsequent fertilization induce the biosynthesis of several hormones, including auxin and gibberellins (GAs), which stimulate fruit set. Circumstantial evidence suggests that the gaseous hormone ethylene may also influence fruit set, but this has yet to be substantiated with molecular or mechanistic data. Here, we examined fruit set at the biochemical and genetic levels, using hormone and inhibitor treatments, and mutants that affect auxin or ethylene signaling. The expression of system-1 ethylene biosynthetic genes and the production of ethylene decreased during pollination-dependent fruit set in wild-type tomato and during pollination-independent fruit set in the auxin hypersensitive mutant iaa9-3. Blocking ethylene perception in emasculated flowers, using either the ethylene-insensitive Sletr1-1 mutation or 1-methylcyclopropene (1-MCP), resulted in elongated parthenocarpic fruit and increased cell expansion, whereas simultaneous treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) inhibited parthenocarpy. Additionally, the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to pollinated ovaries reduced fruit set. Furthermore, Sletr1-1 parthenocarpic fruits did not exhibit increased auxin accumulation, but rather had elevated levels of bioactive GAs, most likely reflecting an increase in transcripts encoding the GA-biosynthetic enzyme SlGA20ox3, as well as a reduction in the levels of transcripts encoding the GA-inactivating enzymes SlGA2ox4 and SlGA2ox5. Taken together, our results suggest that ethylene plays a role in tomato fruit set by suppressing GA metabolism.

CLUMSY VEIN, the Arabidopsis DEAH-box Prp16 ortholog, is required for auxin-mediated development.

Pre-messenger RNA (pre-mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre-mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin-mediated development including male-gametophyte transmission, apical-basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv-1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv-1 mutation does not have an equal influence on pre-mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin-mediated development.

Mesophyll conductance decreases in the wild type but not in an ABA-deficient mutant (aba1) of Nicotiana plumbaginifolia under drought conditions.

Under drought conditions, leaf photosynthesis is limited by the supply of CO2 . Drought induces production of abscisic acid (ABA), and ABA decreases stomatal conductance (gs ). Previous papers reported that the drought stress also causes the decrease in mesophyll conductance (gm ). However, the relationships between ABA content and gm are unclear. We investigated the responses of gm to the leaf ABA content [(ABA)L ] using an ABA-deficient mutant, aba1, and the wild type (WT) of Nicotiana plumbaginifolia. We also measured leaf water potential (ΨL ) because leaf hydraulics may be related to gm . Under drought conditions, gm decreased with the increase in (ABA)L in WT, whereas both (ABA)L and gm were unchanged by the drought treatment in aba1. Exogenously applied ABA decreased gm in both WT and aba1 in a dose-dependent manner. ΨL in WT was decreased by the drought treatment to -0.7 MPa, whereas ΨL in aba1 was around -0.8 MPa even under the well-watered conditions and unchanged by the drought treatment. From these results, we conclude that the increase in (ABA)L is crucial for the decrease in gm under drought conditions. We discuss possible relationships between the decrease in gm and changes in the leaf hydraulics.

Shoot-derived cytokinins systemically regulate root nodulation.

Legumes establish symbiotic associations with nitrogen-fixing bacteria (rhizobia) in root nodules to obtain nitrogen. Legumes control nodule number through long-distance communication between roots and shoots, maintaining the proper symbiotic balance. Rhizobial infection triggers the production of mobile CLE-RS1/2 peptides in Lotus japonicus roots; the perception of the signal by receptor kinase HAR1 in shoots presumably induces the production of an unidentified shoot-derived inhibitor (SDI) that translocates to roots and blocks further nodule development. Here we show that, CLE-RS1/2-HAR1 signalling activates the production of shoot-derived cytokinins, which have an SDI-like capacity to systemically suppress nodulation. In addition, we show that LjIPT3 is involved in nodulation-related cytokinin production in shoots. The expression of LjIPT3 is activated in an HAR1-dependent manner. We further demonstrate shoot-to-root long-distance transport of cytokinin in L. japonicus seedlings. These findings add essential components to our understanding of how legumes control nodulation to balance nutritional requirements and energy status.

Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids.

The notion that plants use specialized metabolism to protect against environmental stresses needs to be experimentally proven by addressing the question of whether stress tolerance by specialized metabolism is directly due to metabolites such as flavonoids. We report that flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana. Metabolome and transcriptome profiling and experiments with oxidative and drought stress in wild-type, single overexpressors of MYB12/PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES1) or MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), double overexpressors of MYB12 and PAP1, transparent testa4 (tt4) as a flavonoid-deficient mutant, and flavonoid-deficient MYB12 or PAP1 overexpressing lines (obtained by crossing tt4 and the individual MYB overexpressor) demonstrated that flavonoid overaccumulation was key to enhanced tolerance to such stresses. Antioxidative activity assays using 2,2-diphenyl-1-picrylhydrazyl, methyl viologen, and 3,3'-diaminobenzidine clearly showed that anthocyanin overaccumulation with strong in vitro antioxidative activity mitigated the accumulation of reactive oxygen species in vivo under oxidative and drought stress. These data confirm the usefulness of flavonoids for enhancing both biotic and abiotic stress tolerance in crops.

Side-chain modification of cytokinins controls shoot growth in Arabidopsis.

Cytokinins (CKs), a class of plant hormones, are central regulators of plant growth and development. Based on numerous physiological and genetic studies, the quantitative regulation of cytokinin levels is the major mechanism regulating cytokinin action in diverse developmental processes. Here, we identified a different mechanism with which the physiological function of CK is modulated through side-chain modification (trans-hydroxylation). The trans-hydroxylation that forms trans-zeatin (tZ)-type CK from N(6)-(Δ(2)-isopentenyl)adenine (iP)-type CK is catalyzed by the cytochrome P450 enzymes CYP735A1 and CYP735A2 in Arabidopsis. Deficiency in trans-hydroxylation activity results in dramatic retardation of shoot growth without affecting total CK quantity, while augmentation of the activity enhances shoot growth. Application of exogenous tZ but not iP recovers the wild-type phenotype in the mutants, indicating that trans-hydroxylation modifies the physiological function of CK. We propose that the control of cytokinin function by side-chain modification is crucial for shoot growth regulation in plants.

Nitrogen-dependent regulation of de novo cytokinin biosynthesis in rice: the role of glutamine metabolism as an additional signal.

Cytokinin activity in plants is closely related to nitrogen availability, and an Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT), IPT3, is regulated by inorganic nitrogen sources in a nitrate-specific manner. In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins. Pre-treatment of roots with l-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while glutamine application induced their expression. Thus, neither nitrate nor ammonium, but glutamine or a related metabolite, is essential for the induction of these IPT genes in rice. On the other hand, glutamine-dependent induction of IPT3 occurs in Arabidopsis, at least to some extent. In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen. We conclude that plants possess multiple regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate growth in response to nitrogen availability.

Agrobacterium tumefaciens tumor morphology root plastid localization and preferential usage of hydroxylated prenyl donor is important for efficient gall formation.

Upon Agrobacterium tumefaciens infection of a host plant, Tumor morphology root (Tmr) a bacterial adenosine phosphate-isopentenyltransferase (IPT), creates a metabolic bypass in the plastid for direct synthesis of trans-zeatin (tZ) with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as the prenyl donor. To understand the biological importance of Tmr function for gall formation, we compared Tmr and Trans-zeatin secretion (Tzs) another agrobacterial IPT that functions within the bacterial cell. Although there is no significant difference in their substrate specificities in vitro, ectopic overexpression of Tzs in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of comparable amounts of tZ- and N6-(Δ²-isopentenyl)adenine (iP)-type cytokinins, whereas overexpression of Tmr resulted exclusively in the accumulation of tZ-type cytokinins. Ectopic expression of Tzs in plant cells yields only small amounts of the polypeptide in plastid-enriched fractions. Obligatory localization of Tzs into Arabidopsis plastid stroma by translational fusions with ferredoxin transit peptide (TP-Tzs) increased the accumulation of both tZ- and iP-type cytokinins. Replacement of tmr on the Ti plasmid with tzs, TP-tzs, or an Arabidopsis plastidic IPT induced the formation of smaller galls than wild-type A. tumefaciens, and they were accompanied by the accumulation of iP-type cytokinins. Tmr is thus specialized for plastid localization and preferential usage of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in vivo and is important for efficient gall formation.