Isopentenyl Transferase Research Articles

Synthesis of vitamin K2 by isopentenyl transferase NovA in Pichia pastoris Gpn12

The effect of methanol addition on the heterologous expression of isoprenyl transferase NovQ was studied in Pichia pastoris Gpn12, with menadione and isopentenol as precursors to catalyze vitamin K2 (MK-3) synthesis. The expression of NovQ increased by 36% when 2% methanol was added every 24 h. The influence of initial pH, temperature, methanol addition, precursors (menadione, isopentenol) addition, catalytic time and cetyltrimethyl-ammonium bromide (CTAB) addition were explored in the P. pastoris whole-cell catalytic synthesis process of MK-3 in shaking flask. Three significant factors were then studied by response surface method. The optimal catalytic conditions obtained were as follows: catalytic temperature 31.56 ℃, menadione 295.54 mg/L, catalytic time 15.87 h. Consistent with the response surface prediction results, the optimized yield of MK-3 reached 98.47 mg/L in shaking flask, 35% higher than that of the control group. On this basis, the production in a 30-L fermenter reached 189.67 mg/L when the cell catalyst of 220 g/L (dry weight) was used to catalyze the synthesis for 24 h. This method laid the foundation for the large-scale production of MK-3 by P. pastoris Gpn12.

Isolation and Functional Analysis of the Regulation of Branching by Isopentenyl Transferase Gene CmIPT1 in <i>Chrysanthemum morifolium </i>cv. ‘Jinba’

Today’s chrysanthemums are highly evolved flowering plants and they are considered as one of the most important ornamental cut flowers. In this research an isopentenyl transferase gene named CmIPT1 was isolated from Chrysanthemum morifolium cv. ‘Jinba’ using RACE and RT-PCR methods. The full cDNA sequence of CmIPT1 was 873 bp which encoded a deduced protein of 290 amino acids. It contained GxxGxGKS which is a conserved sequence of the typical domain of IPT family. The phylogenetic tree analysis of CmIPT1 in Chrysanthemum morifolium cv. ‘Jinba’ shows that it has the closest relationship with CcIPT1 in Cynara cardunculus var. scolymus. Expression of CmIPT1 was higher in stems and apex, whereas it was lower in leaves and roots. And the overexpression of CmIPT1 obviously increased the number of rosette branches in Arabidopsis. Here, in our study, we showed that CmIPT1 is a positive regulator of branch development in Chrysanthemum and may play a key role in regulating lateral branch formation of Chrysanthemum plants.

Genetic engineering of sweet potatoes (Ipomoea batatas) using isopentenyl transferase gene for enhanced drought tolerance

Nawiri SO, Oduor RO, Jalemba AM. 2017. Genetic engineering of sweet potatoes (Ipomoea batatas) using isopentenyl transferase gene for enhanced drought tolerance. Asian J Agric 1: 85-99. Approximately 70% of yield crop reduction worldwide is caused by drought. Due to severe drought which happened many times as a result of climate change, substantial yield deprivation is usual among the major cereals such as maize, wheat, and barley.. Therefore, drought tolerant crops that still yield amidst erratic climatic phenomenon are greatly needed. Due to its capability to produce high yield in a short period, sweet potato is suitable for cultivation in regions with limited or erratic rain water supply where other food crops cannot grow easily. Nevertheless, its sensitivity to water deficit may lead to the adverse crop growth and yield. By conventional hybridization method, sweet potato is tried to be improved, but it gives unsatisfied results due to its high male sterility, sexual incompatibility and hexaploid nature of its genome.The aim of this study, therefore, is to develop new varieties of sweet potato with improved tolerance to water-deficit stress for sustainable production of sweet potato under water-limited conditions. Three sweet potato genotypes: Jewel, Kemb36, and Ksp36 were transformed using isopentenyl transferase gene (IPT) that delays drought-induced senescence via up-regulation of cytokinin biosynthesis, under the control of a waterdeficit responsive and maturation specific promoter (PSARK). The PNOV-IPT gene construct was introduced into sweet potato to evaluate their transformability and regenerability. It is done via Agrobacterium tumefaciens strain EHA101 and the plants subsequently regenerated via somatic embryogenesis. Jewel genotype recorded the highest transformation and regeneration frequency followed by Kemb36 and KSP36. Calli were cultured on media supplemented with various mannose concentrations to evaluate the suitability of mannose as a selectable marker for sweet potato, and it was figured out that 30 g/L concentration was optimal for selection of transformed events. At the time of PCR analysis, Jewel had the highest transformation efficiency followed by Kemb36. At the time for evaluation on drought tolerance under controlled conditions, the sweet potato showed delayed senescence and greater drought tolerance under water deficit conditions in the glasshouse. These plants exhibited better growth, higher yield, higher water status maintenance, higher chlorophyll content, and thus higher photosynthetic rates under reduced water conditions in comparison to wild-type. These results, therefore, indicated that expression of isopentenyl transferase gene in sweet potato significantly improves drought tolerance. Therefore, IPT gene should be used to transform other economically important food crops to delay drought-induced senescence and enhance drought tolerance.

Transcriptional factors for stress signaling, oxidative protection, and protein modification in ipt-transgenic creeping bentgrass exposed to drought stress

Overexpressing the cytokinin (CK)-synthesizing gene, isopentenyl transferase (ipt), is known to elevate endogenous CK levels and positively regulate drought tolerance. The objective of this study was to identify transcriptional factors (TFs) and their down-stream genes responsive to ipt overexpression which are associated with improved drought tolerance in creeping bentgrass (Agrostis stolonifera). Transcriptomic profiling was conducted in both the wild type (WT) (cv. ‘Penncross’) and ipt-transgenic creeping bentgrass exposed to well-watered condition and drought stress. A total of 65 TFs showed significantly increased and 62 TFs decreased expression levels in ipt-transgenic compared to the WT exposed to drought stress. The down-stream genes of 15 TFs were also differentially expressed between ipt-transgenic and WT plants. In addition, ipt-transgenic and WT exposed to drought stress also showed differential expression of genes for receptor kinases, stress defense, protein modification, and protein degradation. The transcriptomic analysis revealed that overexpressing ipt gene caused transcriptional changes with central hubs of bHLH148 and MYB4/4-like and WRKY28/53/71. Such TFs also showed to be associated with the up-regulation of transcripts that are involved in jasmonic acid signaling and down-regulation of abscisic acid signaling and antioxidant response, as well as efficient MAPKKK-mediated protein modification and RNA degradation. The changes in the aforementioned pathways could contribute to the transcriptional regulation of CK for improving drought tolerance in creeping bentgrass.

Cloning and Expression Analysis of a Farnesyl Diphosphate Synthase (FPPS) Gene from Chamaemelum nobile

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene (CnFPPS) was cloned from Chamaemelum nobile. The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile. Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla. The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile.

Cytokinin Metabolism of Pathogenic Fungus Leptosphaeria maculans Involves Isopentenyltransferase, Adenosine Kinase and Cytokinin Oxidase/Dehydrogenase.

Among phytohormones, cytokinins (CKs) play an important role in controlling crucial aspects of plant development. Not only plants but also diverse microorganisms are able to produce phytohormones, including CKs, though knowledge concerning their biosynthesis and metabolism is still limited. In this work we demonstrate that the fungus Leptosphaeria maculans, a hemi-biotrophic pathogen of oilseed rape (Brassica napus), causing one of the most damaging diseases of this crop, is able to modify the CK profile in infected B. napus tissues, as well as produce a wide range of CKs in vitro, with the cis-zeatin derivatives predominating. The endogenous CK spectrum of L. maculans in vitro consists mainly of free CK bases, as opposed to plants, where other CK forms are mostly more abundant. Using functional genomics, enzymatic and feeding assays with CK bases supplied to culture media, we show that L. maculans contains a functional: (i) isopentenyltransferase (IPT) involved in cZ production; (ii) adenosine kinase (AK) involved in phosphorylation of CK ribosides to nucleotides; and (iii) CK-degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Our data further indicate the presence of cis–trans isomerase, zeatin O-glucosyltransferase(s) and N6-(Δ2-isopentenyl)adenine hydroxylating enzyme. Besides, we report on a crucial role of LmAK for L. maculans fitness and virulence. Altogether, in this study we characterize in detail the CK metabolism of the filamentous fungi L. maculans and report its two novel components, the CKX and CK-related AK activities, according to our knowledge for the first time in the fungal kingdom. Based on these findings, we propose a model illustrating CK metabolism pathways in L. maculans.

Light influences cytokinin biosynthesis and sensing in Nostoc (cyanobacteria).

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation.

ABA Biosynthesis Genes are Down-regulated While Auxin and Cytokinin Biosynthesis Genes are Up-regulated During the Release of Grapevine Buds From Endodormancy

Auxin–cytokinin (CK) interactions have been extensively studied in the control of bud outgrowth in herbaceous plants. However, in temperate woody plants where the meristem of dormant buds can be repressed by either exogenous or endogenous factors, abscisic acid (ABA) has been suggested as a potential regulator of bud outgrowth. To investigate the involvement of ABA, CK, and auxin on bud sprouting in Vitis vinifera, single-bud cuttings were used under forced conditions. This artificial bud sprouting system mimics and hastens the natural sprouting process that occurs in spring. Our results showed that expression of the ABA biosynthesis gene VvNCED1 decreased during incubation, whereas expression of the ABA catabolism gene VvA8H3 remained unaltered. Expression of CK biosynthesis-related genes ISOPENTENYL TRANSFERASE (VvIPTs) and LONELY GUY (VvLOG1), CK catabolism-related gene CYTOKININ OXIDASE (VvCKX3), and key auxin biosynthesis gene VvYUC3 increased with incubation time. Moreover, treatment with hydrogen cyanamide (HC), a compound that breaks vine latency, increased expression of VvIPTs and VvLOG1 and reduced expression of VvCKX3 and VvNCED1. These results are consistent with previous reports indicating that HC increases CK levels and decreases ABA levels in grapevine buds. Taken together, the results suggest that in the vine, bud sprouting is preceded by a decrease in ABA content and an increase in CK and auxin levels.

Cytokinin biosynthesis ISOPENTENYLTRANSFERASE genes are differentially expressed during phyllomorph development in the acaulescent Streptocarpus rexii (Gesneriaceae)

The enzyme ISOPENTENYLTRANSFERASE (IPT) is responsible for the rate limiting step of cytokinin biosynthesis, an important plant hormone with key roles in meristem maintenance and organ development. In this study, we isolated IPT genes from the acaulescent Streptocarpus rexii, a plant that shows an unorthodox development starting with post-germination anisocotyly, in which cytokinins play an integral role. Three adenosine phosphate-IPTs and two tRNA-IPTs were isolated from S. rexii. Their expression levels and patterns in different tissues were compared by means of realtime-PCR and mRNA in-situ hybridization. We found that each SrIPT had a distinctive expression pattern. Interestingly, in vegetative tissues as well as in meristems only the adenosine phosphate-IPT SrIPT5 and the tRNA-IPT SrIPT9 were found. In addition, they were differentially affected by external hormone application, suggesting their different regulation and expression during meristem formation and maintenance and lamina growth. Our results indicate that SrIPTs are involved in shaping the architecture of S. rexii, working differentially and redundantly, and show that differentially expressed IPT genes regulate plant form.

Modulating the Levels of Plant Hormone Cytokinins at the Host-Pathogen Interface.

Cytokinins are adenine and non-adenine derived heterogeneous class of regulatory molecules that participate in almost every aspect of plant biology. They also affect plant defense responses as well as help microbial pathogens to establish pathogenesis. The functional approaches that ensure desired and subtle modulations in the levels of plant cytokinins are highly instrumental in assessing their functions in plant immunity. Here, we describe a detailed working protocol regarding the enhanced production of cytokinins from plants that harbor isopentenyltransferase (IPT) enzyme gene under the control of 4xJERE (jasmonic acid and elicitor-responsive element) pathogen-inducible promoter. Our devised expression system is a context-dependent solution when it comes to investigating host-pathogen interactions under the modulated conditions of plant cytokinins.

Differentially Expressed Genes Associated with Improved Drought Tolerance in Creeping Bentgrass Overexpressing a Gene for Cytokinin Biosynthesis.

Transformation with an isopentenyl transferase (ipt) gene controlling cytokinin (CK) synthesis has been shown to enhance plant drought tolerance. The objective of this study was to identify differentially-expressed genes (DEGs) in creeping bentgrass (Agrostis stolonifera) overexpressing ipt compared to non-transgenic plants. The ipt transgene was controlled by a senescence-activated promoter (SAG12). Both a null transformed line (NT) and SAG12-ipt plants were exposed to drought stress in an environmentally-controlled growth chamber until the soil water content declined to approximately 5% and leaf relative water content declined to 47%, which were both significantly below the well-watered controls. RNA was extracted from leaf samples of both well-watered and drought-stressed plants. Eight sets of subtractive hybridizations were performed for detection of up-regulated and down-regulated genes due to the presence of the transgene and due to drought stress in both NT and transgenic plants. Sequencing analysis revealed the identity of 252 DEGs due to either the transgene and drought stress. Sequencing analysis of 170 DEGs identified genes encoding for proteins that were related to energy production, metabolism, stress defense, signaling, protein synthesis and transport, and membrane transport could play major roles in the improved drought tolerance by overexpressing ipt in creeping bentgrass.

Fungal derived cytokinins are necessary for normal Ustilago maydis infection of maize

Phytohormones derived from fungi play a key role in regulating plant–pathogen interactions; however, deciphering the separate contributions of pathogen and plant during infection has been difficult. Here, the Ustilago maydis–Zea mays pathosystem was used to investigate this chemical exchange. Ustilago maydis, the causative agent of maize smut, produces cytokinins (CK), which are a group of phytohormones responsible for directing plant development. The characteristic symptom of smut disease is the formation of tumours composed of plant and fungal tissue. Isopentenyltransferase (IPT) catalyses the rate‐limiting step in CK biosynthesis, and U. maydis strains in which the sole tRNA‐ipt gene was deleted no longer produced CKs. These deletion strains elicited fewer, smaller tumours than the pathogenic strain SG200. High performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI MS/MS) was used to detect and quantify phytohormone levels in infected tissue. This revealed that key hormone changes in SG200 infections were not present in infections by deletion strains, suggesting that CK production by U. maydis is required for the altered phytohormone profile in infected tissue relative to uninfected tissue. Separate analyses indicated that U. maydis tRNA‐ipt mutants might be altered in their ability to metabolize CKs taken up from the environment. Mining the U. maydis genome identified genes encoding putative CK signalling and biosynthesis proteins.

The human tRNA-modifying protein, TRIT1, forms amyloid fibers in vitro

TRIT1 is a highly conserved tRNA isopentenyl transferase that modifies a subset of tRNAs in human cells and is a candidate tumor suppressor in lung cancer in certain ethnic populations. The yeast homologue, Mod5, has similar tRNA-modifying functions in the cytoplasm and is required for the transcriptional silencing activity of RNA polymerase II promoters near tRNA genes in the nucleus, a phenomenon termed tRNA gene mediated (tgm) silencing. Furthermore, Mod5 can fold into amyloid fibers in vitro and in vivo, which confers resistance to certain fungicides in yeast. Since TRIT1 complements both tRNA modifying and tgm-silencing activities in yeast where the Mod5 gene has been deleted, it seemed possible that TRIT1 might also have amyloid-forming capabilities. Here we show that TRIT1, like Mod5, directly binds to tRNAs that are both substrate and non-substrates for modification with similar affinity, and to an unstructured, non-tRNA. Binding appears to involve distinct protein-RNA multimers which decrease in electrophoretic mobility as the protein to RNA ratio increases. Furthermore, we characterize TRIT1 as a novel human amyloid fiber forming protein. We discuss these data in light of TRIT1's functional roles and possible implications for disease.

English

Maize is a staple food crop for millions of Africans. Despite this fact, African farmers have been harvesting average grain yield of not more than 2 t/ha while there is a potential of producing more than 10 t/ha. Drought is one of the major abiotic constraints contributing to this low productivity. Drought diminishes crop productivity mainly by causing premature leaf senescence. The ipt gene codes for isopentenyltransferase (IPT) enzyme which catalyzes the rate limiting step in the biosynthesis of cytokinin and has been shown to enhance tolerance to drought in transgenic crops by delaying drought-induced leaf senescence. This created interest to investigate if ipt gene can be useful in enhancing drought tolerance in locally adapted African tropical maize genotypes. The tropical maize inbred line CML216 was transformed with ipt gene using Agrobacterium-mediated transformation method. Five transgenic lines which were proved to be stably transformed through Southern blot analysis with copy number of 2 to 4 per event were developed. In drought assay carried out in the glass house, transgenic lines expressing the ipt gene showed tolerance to drought as revealed by delayed leaf senescence compared to the wild type plants. Transgenic plants maintained higher relative water content and total chlorophyll during the drought period and produced significantly higher mean grain yield of 44.3 g/plant while the wild type plants produced mean grain yield of 1.43 g/plant. It is proposed that the transgenic lines developed in this study can be further tested for tolerance to drought under contained field trials. Furthermore, transgenic lines developed can be used in breeding programs to improve drought tolerance in other commercial tropical maize genotypes through conventional breeding. Key words: Cytokinin, delayed leaf senescence, drought inducible, CML216, ipt gene.

Identification and characterization of miRNAs involved in adventitious branches formation of Gracilaria lichenoides in vitro

The main morphological response of Gracilaria lichenoides explants to wounding in vitro was the direct production of adventitious branches (ABs) within a short period rather than callus formation, similar to other seaweeds. However, the underlying mechanism remains unclear because of the lack of genome information. MicroRNAs (miRNAs) play a role in plant growth and development by targeting mRNAs for translational repression or cleavage. Here, we investigated small RNA-mediated regulation of ABs formation by examining the post-transcriptional profiles of different treatment samples of G. lichenoides using the Illumina sequencing platform. The results showed that miR156g-3p, miR2597, miR3447-3p, miR169j-3p and other miRNAs families play significant roles in ABs formation in G. lichenoides through their predicted targets, including phospholipase D (PLD), auxin response factor (ARF), protein phosphatase 2C (PP2C), CTR1 and adenylate isopentenyltransferase (IPT). In addition, we identified 30 putative unique Gracilariales miRNAs in two small RNA libraries that may be involved in ABs induction. Profiling of miRNAs and their target genes of G. lichenoides provide new insight on miRNAs networks involved in ABs formation in seaweed.

Analysis the role of arabidopsis CKRC6/ASA1 in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the a-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.

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.

Toxicity and bio-effects of CuO nanoparticles on transgenic Ipt-cotton

ABSTRACTThis study investigated the effects of copper oxide nanoparticles (CuO NPs) on the growth and development of transgenic cotton harboring the Ipt gene, which encodes isopentenyl transferase (Ipt). Three concentrations of CuO NPs were evaluated: 10, 200, and 1000 mg·L-1, each with three replicates. The height and the root length were 26.91% and 42.80% decreased after 10-day exposure with 1000 mg·L-1 CuO NPs, respectively.In addition, less abundant on root hairs and lower in shoot biomass of Ipt-cotton when compared with the control group. The growth of Ipt-cotton was not affected by 10 mg·L-1 CuO NPs, but a high concentration of CuO NPs promoted the absorption of Fe and Na into roots, and inhibited the production of phytohormones in Ipt-cotton. The CuO NPs increased the concentration of iPA in shoots, which can delay senescence. The extent of the increase in iPA in response to CuO NPs should be relative to the amount of Ipt immobilized onto the NPs in the plant tissue. To our knowledge, this is the first study to evaluate the phytotoxicity of CuO NPs to Ipt-transgenic cotton. These results establish a baseline for further research on the effects of nanoparticles on transgenic crops harboring the Ipt gene.

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.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.