Expression Of Isopentenyl Transferase Research Articles

Stress-induced expression of IPT gene in transgenic wheat reduces grain yield penalty under drought

BackgroundThe heterologous expression of isopentenyl transferase (IPT) under the transcriptional control of the senescence-associated receptor-like kinase (SARK) promoter delayed cellular senescence and, through it, increased drought tolerance in plants. To evaluate the effect of pSARK::IPT expression in bread wheat, six independent transgenic events were obtained through the biolistic method and evaluated transgene expression, phenology, grain yield and physiological biomass components in plants grown under both drought and well-irrigating conditions. Experiments were performed at different levels: (i) pots and (ii) microplots inside a biosafety greenhouse, as well as under (iii) field conditions. ResultsTwo transgenic events, called TR1 and TR4, outperformed the wild-type control under drought conditions. Transgenic plants showed higher yield under both greenhouse and field conditions, which was positively correlated to grain number (given by more spikes and grains per spike) than wild type. Interestingly, this yield advantage of the transgenic events was observed under both drought and well-watered conditions. ConclusionsThe results obtained allow us to conclude that the SARK promoter-regulated expression of the IPT gene in bread wheat not only reduced the yield penalty produced by water stress but also led to improved productivity under well-watered conditions.

Cytokinin glucosyl transferases, key regulators of cytokinin homeostasis, have potential value for wheat improvement.

SummaryThe cytokinins, which are N6‐substituted adenine derivatives, control key aspects of crop productivity. Cytokinin levels are controlled via biosynthesis by isopentenyl transferase (IPT), destruction by cytokinin oxidase/dehydrogenase (CKX), and inactivation via glucosylation by cytokinin glucosyl transferases (CGTs). While both yield components and tolerance to drought and related abiotic stressors have been positively addressed via manipulation of IPT and/or CKX expression, much less attention has been paid to the CGTs. As naming of the CGTs has been unclear, we suggest COGT, CNGT, CONGT and CNOGT to describe the O‐, N‐ and dual function CGTs. As specific CGT mutants of both rice and arabidopsis showed impacts on yield components, we interrogated the wheat genome database, IWGSC RefSeq v1.0 & v2.0, to investigate wheat CGTs. Besides providing unambiguous names for the 53 wheat CGTs, we show their expression patterns in 70 developmental tissues and their response characteristics to various stress conditions by reviewing more than 1000 RNA‐seq data sets. These revealed various patterns of responses and showed expression generally being more limited in reproductive tissues than in vegetative tissues. Multiple cis‐regulatory elements are present in the 3 kb upstream of the start codons of the 53 CGTs. Elements associated with abscisic acid, light and methyl jasmonate are particularly over‐represented, indicative of the responsiveness of CGTs to the environment. These data sets indicate that CGTs have potential value for wheat improvement and that these could be targeted in TILLING or gene editing wheat breeding programmes.

The effect of auxin and strigolactone on ATP/ADP isopentenyltransferase expression and the regulation of apical dominance in peach

We confirmed the roles of auxin, CK, and strigolactones in apical dominance in peach and established a model of plant hormonal control of apical dominance in peach. Auxin, cytokinin, and strigolactone play important roles in apical dominance. In this study, we analyzed the effect of auxin and strigolactone on the expression of ATP/ADP isopentenyltransferase (IPT) genes (key cytokinin biosynthesis genes) and the regulation of apical dominance in peach. After decapitation, the expression levels of PpIPT1, PpIPT3, and PpIPT5a in nodal stems sharply increased. This observation is consistent with the changes in tZ-type and iP-type cytokinin levels in nodal stems and axillary buds observed after treatment; these changes are required to promote the outgrowth of axillary buds in peach. These results suggest that ATP/ADP PpIPT genes in nodal stems are key genes for cytokinin biosynthesis, as they promote the outgrowth of axillary buds. We also found that auxin and strigolactone inhibited the outgrowth of axillary buds. After decapitation, IAA treatment inhibited the expression of ATP/ADP PpIPTs in nodal stems to impede the increase in cytokinin levels. By contrast, after GR24 (GR24 strigolactone) treatment, the expression of ATP/ADP IPT genes and cytokinin levels still increased markedly, but the rate of increase in gene expression was markedly lower than that observed after decapitation in the absence of IAA (indole-3-acetic acid) treatment. In addition, GR24 inhibited basipetal auxin transport at the nodes (by limiting the expression of PpPIN1a in nodal stems), thereby inhibiting ATP/ADP PpIPT expression in nodal stems. Therefore, strigolactone inhibits the outgrowth of axillary buds in peach only when terminal buds are present.

AUXIN RESPONSE FACTOR3 Regulates Floral Meristem Determinacy by Repressing Cytokinin Biosynthesis and Signaling.

Successful floral meristem (FM) determinacy is critical for subsequent reproductive development and the plant life cycle. Although the phytohormones cytokinin and auxin interact to coregulate many aspects of plant development, whether and how cytokinin and auxin function in FM determinacy remain unclear. Here, we show that in Arabidopsis thaliana, cytokinin homeostasis is critical for FM determinacy. In this developmental context, auxin promotes the expression of AUXIN RESPONSE FACTOR3 (ARF3) to repress cytokinin activity. ARF3 directly represses the expression of ISOPENTENYLTRANSFERASE (IPT) family genes and indirectly represses LONELY GUY (LOG) family genes, both of which encode enzymes required for cytokinin biosynthesis. ARF3 also directly inhibits the expression of ARABIDOPSIS HISTIDINE KINASE4, a cytokinin receptor gene, resulting in reduced cytokinin activity. Consequently, ARF3 controls cell division by regulating cell cycle gene expression through cytokinin. In flowers, we show that AGAMOUS (AG) dynamically regulates the expression of ARF3 and IPTs, resulting in coordinated regulation of FM maintenance and termination through cell division. Moreover, genome-wide transcriptional profiling revealed both repressive and active roles for ARF3 in early flower development. Our findings establish a molecular link between AG and auxin/cytokinin and shed light on the mechanisms of stem cell maintenance and termination in the FM.

Water deficit stress tolerance in maize conferred by expression of an isopentenyltransferase (IPT) gene driven by a stress- and maturation-induced promoter

Senescence can be delayed in transgenic plants overexpressing the enzyme isopentenyltransferase (IPT) due to stress-induced increased levels of endogenous cytokinins. This trait leads to sustained photosynthetic activity and improved tolerance to abiotic stress. The aim of this study was to generate and characterize transgenic plants of maize (Zea mays L.) transformed with the IPT gene sequence under the regulation of SARK promoter (protein kinase receptor-associated senescence). Three independent transgenic events and their segregating null controls were evaluated in two watering regimes (WW: well watered; WD: water deficit) imposed for two weeks around anthesis. Our results show that the WD treatment induced IPT expression with the concomitant increase in cytokinin levels, which prolonged the persistence of total green leaf area, and maintained normal photosynthetic rate and stomatal conductance. These trends were accompanied by a minor decrease in number of grains per plant, individual grain weight and plant grain yield as compared to WW plants. Plants expressing the IPT gene under WD had PGR, anthesis and silking dates and biomass levels similar to WW plants. Our results demonstrate that expression of the IPT gene under the regulation of the SARK promoter helps improve productivity under WD conditions in C4 plants like maize.

Cytokinin: a key driver of seed yield.

The cytokinins have been implicated in many facets of plant growth and development including cell division and differentiation, shoot and root growth, apical dominance, senescence, fruit and seed development, and the response to biotic and abiotic stressors. Cytokinin levels are regulated by a balance between biosynthesis [isopentenyl transferase (IPT)], activation [Lonely Guy (LOG)], inactivation (O-glucosyl transferase), re-activation (β-glucosidase), and degradation [cytokinin oxidase/dehydrogenase (CKX)]. During senescence, the levels of active cytokinins decrease, with premature senescence leading to a decrease in yield. During the early stages of fruit and seed development, cytokinin levels are transiently elevated, and coincide with nuclear and cell divisions which are a determinant of final seed size. Exogenous application of cytokinin, ectopic expression of IPT, or down-regulation of CKX have, on occasions, led to increased seed yield, leading to the suggestion that cytokinin may be limiting yield. However, manipulation of cytokinins is complex, not only because of their pleiotropic nature but also because the genes coding for biosynthesis and metabolism belong to multigene families, the members of which are themselves spatially and temporally differentiated. Previous research on yield of rice showed that plant breeders could directly target the cytokinins. Modern genome editing tools could be employed to target and manipulate cytokinin levels to increase seed yield with the concurrent aim of maintaining quality. However, how the cytokinin level is modified and whether IPT or CKX is targeted may depend on whether the plant is considered to be in a source-limiting environment or to be sink limited.

PSARK::IPT expression causes protection of photosynthesis in tobacco plants during N deficiency

Wild type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene coding the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. WT tobacco plants under N deficit showed a drastic reduction in photosynthetic rate (A) and the maximum carboxylation rate of Rubisco (Vcmax), the maximum rate of the electron transport (Jmax) and the use of triose-P (TPU) parameters. However, the expression an IPT gene driven by PSARK, a stress-and maturation-induced promoter, in tobacco plants under N deficiency leads the maintenance of photosynthesis and leaf biomass related with the maintenance of sm-Rubisco transcripts levels. Limited increase in sugar concentration as well as the maintenance of leaf biomass observed in transgenic plants would indicate CKs may play a role in the sink limitation caused by deficiency of N, enhancing the strength of the sink, such as young leaves, and enhancing foliar biomass under severe N deficiency.

An Isopentyl Transferase Gene Driven by the Stress-Inducible rd29A Promoter Improves Salinity Stress Tolerance in Transgenic Tobacco

Soil salinity is a serious worldwide problem. To improve the salt tolerance of plants, an increasing number of genes related to abiotic stress have been recently expressed by genetic engineers. In the present study, the successful introduction into tobacco of isopentenyl transferase (IPT) from Agrobacterium tumefaciens via Agrobacterium-mediated transformation is reported. A stress-inducible genetic construct was cloned using IPT under the control of the stress-inducible promoter rd29A from Arabidopsis thaliana. A total of 40 putative transgenic plant lines were obtained from independent Kan-resistant shoots. IPT integration into the tobacco genome was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Four positive transgenic lines each with a single T-DNA insertion were obtained. Real-time PCR confirmed a marked increase in IPT expression in young tobacco plants harboring rd29A-IPT after short-term exposure to salt. Ectopic IPT overexpression IPT under the control of the stress-inducible rd29A promoter resulted in significantly enhanced tolerance to salt stress. No obvious adverse effect on growth and development was observed in transgenic plants. Two IPT transgenic lines, T10 and T25, were chosen for further physiological analyses. The leaves of transgenic tobacco plants showed significantly prolonged chlorophyll retention times under a 2-week salt-stress treatment (150 mmol L−1). In contrast, the leaves of the non-transformed plants (wild type) gradually senesced under the same condition. After re-watering for 2 weeks, chlorophyll in transgenic plants increased to a level comparable with that in the unstressed plants. On the other hand, the level in the non-transgenic control still remained low. Malondialdehyde (MDA) levels increased in both transgenic plants and the control after salt stress. However, the MDA levels only mildly increased in transgenic plants, and dramatically increased in the control. After re-watering for 7 days, MDA in transgenic plants returned to normal, whereas the level in the control remained high. Superoxide dismutase activity also similarly increased in transgenic plants during salt stress, and returned to normal after re-watering. These results indicate that enhanced reactive oxygen species scavenging capability may play a significant role in acquiring tolerance to abiotic stress.

Regulated Expression of an Isopentenyltransferase Gene (IPT) in Peanut Significantly Improves Drought Tolerance and Increases Yield Under Field Conditions

Isopentenyltransferase (IPT) is a critical enzyme in the cytokinin biosynthetic pathway. The expression of IPT under the control of a maturation- and stress-induced promoter was shown to delay stress-induced plant senescence that resulted in an enhanced drought tolerance in both monocot and dicot plants. This report extends the earlier findings in tobacco and rice to peanut (Arachis hypogaea L.), an important oil crop and protein source. Regulated expression of IPT in peanut significantly improved drought tolerance in both laboratory and field conditions. Transgenic peanut plants maintained higher photosynthetic rates, higher stomatal conductance and higher transpiration than wild-type control plants under reduced irrigation conditions. More importantly, transgenic peanut plants produced significantly higher yields than wild-type control plants in the field, indicating a great potential for the development of crops with improved performance and yield in water-limited areas of the world.

Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence-associated genes SAG12 or SAG13 (P(SAG12)::IPT and P(SAG13)::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double-transgenic tomato plants expressing both AtHXK1 and either P(SAG12)::IPT or P(SAG13)::IPT. We found that expression of IPT in the double-transgenic plants could not prevent the accelerated senescence induced by over-expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria-associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing P(SAG12)::IPT and P(SAG13)::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.

Reactivation of Meristem Activity and Sprout Growth in Potato Tubers Require Both Cytokinin and Gibberellin

Reactivation of dormant meristems is of central importance for plant fitness and survival. Due to their large meristem size, potato (Solanum tuberosum) tubers serve as a model system to study the underlying molecular processes. The phytohormones cytokinins (CK) and gibberellins (GA) play important roles in releasing potato tuber dormancy and promoting sprouting, but their mode of action in these processes is still obscure. Here, we established an in vitro assay using excised tuber buds to study the dormancy-releasing capacity of GA and CK and show that application of gibberellic acid (GA(3)) is sufficient to induce sprouting. In contrast, treatment with 6-benzylaminopurine induced bud break but did not support further sprout growth unless GA(3) was administered additionally. Transgenic potato plants expressing Arabidopsis (Arabidopsis thaliana) GA 20-oxidase or GA 2-oxidase to modify endogenous GA levels showed the expected phenotypical changes as well as slight effects on tuber sprouting. The isopentenyltransferase (IPT) from Agrobacterium tumefaciens and the Arabidopsis cytokinin oxidase/dehydrogenase1 (CKX) were exploited to modify the amounts of CK in transgenic potato plants. IPT expression promoted earlier sprouting in vitro. Strikingly, CKX-expressing tubers exhibited a prolonged dormancy period and did not respond to GA(3). This supports an essential role of CK in terminating tuber dormancy and indicates that GA is not sufficient to break dormancy in the absence of CK. GA(3)-treated wild-type and CKX-expressing tuber buds were subjected to a transcriptome analysis that revealed transcriptional changes in several functional groups, including cell wall metabolism, cell cycle, and auxin and ethylene signaling, denoting events associated with the reactivation of dormant meristems.

Growth, yield and shelf-life of isopentenyltransferase (ipt)-gene transformed broccoli

Loss of chlorophyll leading to floret yellowing limits the post-harvest lifespan of broccoli (Brassica oleracea L. var. italica Plenck). Cytokinins are known to delay floral yellowing of plants. A transgene construct pSG766A, which results in the expression of isopentenyltransferase (ipt), the key enzyme for cytokinin synthesis, has been developed in broccoli. Expression of the ipt transgene is triggered by the senescence-associated gene promoter (SAG-13). Three selfed T5 lines of ipt transformed broccoli (lines 101, 102 and 103) have been obtained through selection for single copy insertion, acceptable horticultural traits and transgene ipt activity. These three transgenic inbred lines were evaluated in the field during 2004-2007 to determine their growth, yield and shelf-life after harvest, relative to a non-transgenic inbred line (104) and the parental variety Green King. For most of the vegetative growth parameters measured, year-to-year variability exceeded line-to-line variability. Inbreeding had little impact on the appearance or yield potential of the broccoli lines. Head yields of the transgenic inbred lines 102 and 103 were comparable to the parental variety Green King, but were significantly higher than the non-transgenic inbred line 104, as lines 102 and 103 produced more plants with heavier flower heads. Cytokinin content in the form of isopentenyladenosine was relatively higher in the transgenic lines than in the two non-transgenic controls. When flower heads were stored at 25 ± 2°C, the period required to cause 50% floret yellowing was 7.5 and 8.5 d for the transgenic lines 102 and 103, respectively, compared with 5.6 d for the non-transgenic line 104, and 5.1 d for the parental variety Green King. This study showed that the ipt-transformed inbred lines of broccoli combined acceptable appearance and yields with enhanced shelf-life.Key words: Brassica oleracea L. var. italica Plenck, transgenic broccoli, isopentenyltransferase gene, genetic characterization, shelf-life

Cytokinin-Dependent Photorespiration and the Protection of Photosynthesis during Water Deficit

We investigated the effects of P(SARK)IPT (for Senescence-Associated Receptor KinaseIsopentenyltransferase) expression and cytokinin production on several aspects of photosynthesis in transgenic tobacco (Nicotiana tabacum cv SR1) plants grown under optimal or restricted (30% of optimal) watering regimes. There were no significant differences in stomatal conductance between leaves from wild-type and transgenic P(SARK)-IPT plants grown under optimal or restricted watering. On the other hand, there was a significant reduction in the maximum rate of electron transport as well as the use of triose-phosphates only in wild-type plants during growth under restricted watering, indicating a biochemical control of photosynthesis during growth under water deficit. During water deficit conditions, the transgenic plants displayed an increase in catalase inside peroxisomes, maintained a physical association among chloroplasts, peroxisomes, and mitochondria, and increased the CO(2) compensation point, indicating the cytokinin-mediated occurrence of photorespiration in the transgenic plants. The contribution of photorespiration to the tolerance of transgenic plants to water deficit was also supported by the increase in transcripts coding for enzymes involved in the conversion of glycolate to ribulose-1,5-bisphosphate. Moreover, the increase in transcripts indicated a cytokinin-induced elevation in photorespiration, suggesting the contribution of photorespiration in the protection of photosynthetic processes and its beneficial role during water stress.

Evaluation of selection strategies alternative to nptII in genetic transformation of citrus

The neomycin phosphotransferase (nptII) selection system has proved successful in citrus transformation; however, it may be recommendable to replace it given the pressure exerted against antibiotic-resistance selectable marker genes in transgenic plants. The present work investigates three different selection alternatives, comparing them to nptII selection in two citrus genotypes, Carrizo citrange and Pineapple sweet orange. The first method used the beta-glucuronidase (uidA) reporter marker gene for selection; the second attempted to generate marker-free plants by transforming explants with a multi-auto-transformation (MAT) vector, combining an inducible R/RS-specific recombination system with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes; while the third exploited the phosphomannose isomerase (PMI)/mannose conditional positive selection system. Firstly, GUS screening of all regenerated shoots in kanamycin-free medium gave 4.3% transformation efficiency for both genotypes. Secondly, workable transformation efficiencies were also achieved with the MAT system, 7.2% for citrange and 6.7% for sweet orange. This system affords an additional advantage as it enables selectable marker genes to be used during the in vitro culture phase and later removed from the transgenic plants by inducible recombination and site-specific excision. Thirdly, the highest transformation rates were obtained with the PMI/mannose system, 30% for citrange and 13% for sweet orange, which indicates that this marker is also an excellent candidate for citrus transformation.

Botrytis cinerea induces senescence and is inhibited by autoregulated expression of the IPT gene

Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including Arabidopsis and tomato. Since senescing leaves are particularly susceptible to infection by B. cinerea, we hypothesized that the fungus might induce senescence as part of its mode of action and that delaying leaf senescence might reduce the severity of B. cinerea infections. To examine these hypotheses, we followed the expression of Arabidopsis SAG12, a senescence-specific gene, upon infection with B. cinerea. Expression of SAG12 is induced by B. cinerea infection, indicating that B. cinerea induces senescence. The promoter of SAG12, as well as that of a second Arabidopsis senescence-associated gene, SAG13, whose expression is not specific to senescence, were previously analyzed in tomato plants and were found to be expressed in a similar manner in the two species. These promoters were previously used in tomato plants to drive the expression of isopentenyl transferase (IPT) from Agrobacterium to suppress leaf senescence (Swartzberg et al. in Plant Biology 8:579–586, 2006). In this study, we examined the expression of these promoters following infection of tomato plants with B. cinerea. Both promoters exhibit high expression levels upon B. cinerea infection of non-senescing leaves of tomato plants, supporting our conclusion that B. cinerea induces senescence as part of its mode of action. In contrast to B. cinerea, Trichoderma harzianum T39, a saprophytic fungus that is used as a biocontrol agent against B. cinerea, induces expression of SAG13 only. Expression of IPT, under the control of the SAG12 and SAG13 promoters in response to infection with B. cinerea resulted in suppression of B. cinerea-induced disease symptoms, substantiating our prediction that delaying leaf senescence might reduce susceptibility to B. cinerea.

469 Multi-faceted Approaches to Genetic Engineering of Petunia × hybrida for Delayed Leaf Senescence

In an effort to modify and study leaf senescence, we have produced several different transgenic petunia lines with altered leaf senescence phenotypes. Using two promoters from senescence-associated genes (sag12 & sag13) fused to the isopentenyl transferase (IPT) gene, which catalyzes the rate-limiting step of cytokinin production, we have produced transgenic petunia plants with delayed lower leaf senescence. We have observed that apparent “leaky” expression of IPT gives rise to plants with other morphological alterations such as increased branching habit and decreased root formation. Plants with delayed leaf senescence phenotypes were selected and bred to produce progeny that were evaluated in greenhouse experiments. Breeding characteristics, horticultural performance and reproduction of these plants will be discussed in terms of potential commercial benefits and limitations. Using the sag12 promoter to drive expression of the knotted (KN1) gene, we have also been able to engineer petunia plants with delayed lower leaf senescence. Initial progeny evaluations of sag12-KN1 petunias will also be discussed.