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1.  Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis 
Journal of Experimental Botany  2013;64(14):4239-4253.
Rice MADS29 has recently been reported to cause programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development. However, analyses involving OsMADS29 protein expression domains and characterization of OsMADS29 gain-of-function and knockdown phenotypes revealed novel aspects of its function in maintaining hormone homeostasis, which may have a role in the development of embryo and plastid differentiation and starch filling in endosperm cells. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signalling and biosynthesis.
doi:10.1093/jxb/ert231
PMCID: PMC3808311  PMID: 23929654
Cytokinin; embryo; endosperm; MADS box; Oryza sativa; starch; seed development.
2.  Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis 
Journal of Experimental Botany  2014;65(9):2533-2543.
Summary
Is there more than one pathway for cytokinin biosynthesis in Physcomitrella? Despite the apparent absence of adenylate-isopentenyltransferases, characterization of ipt1 knockout mutants points towards a second, tRNA-independent cytokinin biosynthesis pathway.
The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N 6-isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.
doi:10.1093/jxb/eru142
PMCID: PMC4036517  PMID: 24692654
Bryophyte; cytokinin; isopentenyladenosine; isopentenyltransferases; moss; tRNA.
3.  A non-synonymous SNP within the isopentenyl transferase 2 locus is associated with kernel weight in Chinese maize inbreds (Zea mays L.) 
BMC Plant Biology  2013;13:98.
Background
Kernel weight, controlled by quantitative trait loci (QTL), is an important component of grain yield in maize. Cytokinins (CKs) participate in determining grain morphology and final grain yield in crops. ZmIPT2, which is expressed mainly in the basal transfer cell layer, endosperm, and embryo during maize kernel development, encodes an isopentenyl transferase (IPT) that is involved in CK biosynthesis.
Results
The coding region of ZmIPT2 was sequenced across a panel of 175 maize inbred lines that are currently used in Chinese maize breeding programs. Only 16 single nucleotide polymorphisms (SNPs) and seven haplotypes were detected among these inbred lines. Nucleotide diversity (π) within the ZmIPT2 window and coding region were 0.347 and 0.0047, respectively, and they were significantly lower than the mean nucleotide diversity value of 0.372 for maize Chromosome 2 (P < 0.01). Association mapping revealed that a single nucleotide change from cytosine (C) to thymine (T) in the ZmIPT2 coding region, which converted a proline residue into a serine residue, was significantly associated with hundred kernel weight (HKW) in three environments (P <0.05), and explained 4.76% of the total phenotypic variation. In vitro characterization suggests that the dimethylallyl diphospate (DMAPP) IPT activity of ZmIPT2-T is higher than that of ZmIPT2-C, as the amounts of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) consumed by ZmIPT2-T were 5.48-, 2.70-, and 1.87-fold, respectively, greater than those consumed by ZmIPT2-C. The effects of artificial selection on the ZmIPT2 coding region were evaluated using Tajima’s D tests across six subgroups of Chinese maize germplasm, with the most frequent favorable allele identified in subgroup PB (Partner B).
Conclusions
These results showed that ZmIPT2, which is associated with kernel weight, was subjected to artificial selection during the maize breeding process. ZmIPT2-T had higher IPT activity than ZmIPT2-C, and this favorable allele for kernel weight could be used in molecular marker-assisted selection for improvement of grain yield components in Chinese maize breeding programs.
doi:10.1186/1471-2229-13-98
PMCID: PMC3704264  PMID: 23826856
Maize; Isopentenyl transferase 2; Association mapping; Artificial selection
4.  A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1) seed mutant and its wild type Mn1 in maize 
In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL). As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1) mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs. Mn1 wild type) using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed >20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis/transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.
doi:10.3389/fpls.2014.00063
PMCID: PMC3935489  PMID: 24616729
seed development; gene expression; sugar methabolism; transfer cells; maize
5.  The role of cytokinin in ovule development in Arabidopsis 
Plant Signaling & Behavior  2013;8(3):e23393.
The life cycle of higher plants alternates between the haploid gametophyte and diploid sporophyte. The female gametophyte (FG), surrounded by the sporophyte, develops within the ovule and orients along the chalazal/micropylar axis. This polarity is important in cell specification and development for both the ovule and FG. Previously, cytokinin was shown to act in the sporophytic tissue to regulate FG development.1,2 In the highlighted study,3 we further showed that enriched cytokinin signaling in chalaza, the central domain of the ovule, is required for the specification of the functional megaspore, which usually occurs in the chalazal-most megaspore after meiosis. The restricted cytokinin signaling in the chalaza is achieved by localized cytokinin biosynthesis and perception. Here, we discuss the implications of this and other studies for the understanding of the role of two-component signaling in FG development and the genetic and cellular interactions between gametophytic and sporophytic cells. Further, we show that cytokinin-deficient mutants display distorted cell morphology in the inner integument and elevated mitotic activity in the maternal sporophyte. These results suggest that cytokinin negatively regulates cell proliferation in the sporophytic tissues surrounding the developing FG, consistent with previous results indicating that cytokinin deficiency causes an increase in the number of cells in the embryos and consequently an enlarged seed size.
doi:10.4161/psb.23393
PMCID: PMC3676506  PMID: 23333968
cytokinin; two-component signaling; female gametophyte; functional megaspore; integument
6.  Roles of DgBRC1 in Regulation of Lateral Branching in Chrysanthemum (Dendranthema ×grandiflora cv. Jinba) 
PLoS ONE  2013;8(4):e61717.
The diverse plasticity of plant architecture is largely determined by shoot branching. Shoot branching is an event regulated by multiple environmental, developmental and hormonal stimuli through triggering lateral bud response. After perceiving these signals, the lateral buds will respond and make a decision on whether to grow out. TCP transcriptional factors, BRC1/TB1/FC1, were previously proven to be involved in local inhibition of shoot branching in Arabidopsis, pea, tomato, maize and rice. To investigate the function of BRC1, we isolated the BRC1 homolog from chrysanthemum. There were two transcripts of DgBRC1 coming from two alleles in one locus, both of which complemented the multiple branches phenotype of Arabidopsis brc1-1, indicating that both are functionally conserved. DgBRC1 was mainly expressed in dormant axillary buds, and down-regulated at the bud activation stage, and up-regulated by higher planting densities. DgBRC1 transcripts could respond to apical auxin supply and polar auxin transport. Moreover, we found that the acropetal cytokinin stream promoted branch outgrowth whether or not apical auxin was present. Basipetal cytokinin promoted outgrowth of branches in the absence of apical auxin, while strengthening the inhibitory effects on lower buds in the presence of apical auxin. The influence of auxin and strigolactons (SLs) on the production of cytokinin was investigated, we found that auxin locally down-regulated biosynthesis of cytokinin in nodes, SLs also down-regulated the biosynthesis of cytokinin, the interactions among these phytohormones need further investigation.
doi:10.1371/journal.pone.0061717
PMCID: PMC3629106  PMID: 23613914
7.  AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis 
PLoS ONE  2009;4(12):e8236.
Background
Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited.
Methodology/Principal Findings
Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2's overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N6-(Δ2-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays.
Conclusions/Significance
Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.
doi:10.1371/journal.pone.0008236
PMCID: PMC2785485  PMID: 20011053
8.  Accumulation of cytokinins in roots and their export to the shoots of durum wheat plants treated with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) 
Journal of Experimental Botany  2014;65(9):2287-2294.
Cytokinin flow from roots to shoots can serve as a long-distance signal important for root-to-shoot communication. In the past, changes in cytokinin flow from roots to shoots have been mainly attributed to changes in the rate of synthesis or breakdown in the roots. The present research tested the possibility that active uptake of cytokinin by root cells may also influence its export to shoots. To this end, we collapsed the proton gradient across root membranes using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit secondary active uptake of exogenous and endogenous cytokinins. We report the impact of CCCP on cytokinin concentrations and delivery in xylem sap and on accumulation in shoots of 7-day-old wheat plants in the presence and absence of exogenous cytokinin applied as zeatin. Zeatin treatment increased the total accumulation of cytokinin in roots and shoots but the effect was smaller for the shoots. Immunohistochemical localization of cytokinins using zeatin-specific antibodies showed an increase in immunostaining of the cells adjacent to xylem in the roots of zeatin-treated plants. Inhibition of secondary active cytokinin uptake by CCCP application decreased cytokinin accumulation in root cells but increased both flow from the roots and accumulation in the shoots. The possible importance of secondary active uptake of cytokinins by root cells for the control of their export to the shoot is discussed.
doi:10.1093/jxb/eru113
PMCID: PMC4036502  PMID: 24692646
Cytokinin; protonophore; root-to-shoot communication; secondary active uptake; Triticum durum; xylem transport; zeatin.
9.  Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants 
Journal of Experimental Botany  2010;62(1):125-140.
Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K+ (20%) and decreases in the toxic ion Na+ (by 30%) and abscisic acid (by 20–40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.
doi:10.1093/jxb/erq266
PMCID: PMC2993914  PMID: 20959628
ABA; cytokinins; grafting; IPT; root zone temperature; root to shoot signalling; salinity; Solanum lycopersicum
10.  HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes 
BMC Plant Biology  2012;12:206.
Background
CKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium.
Results
The mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T1 lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T0 plants and T1 lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes.
The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T1 lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively affected kernel germination or anther development and as a consequence setting the seeds. The final phenotypic effect was the decreased productivity of T0 plants and T1 lines obtained via the biolistic silencing of HvCKX2.
Conclusion
The phenotypic result, which was higher productivity of silenced lines obtained via Agrobacterium, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of Agrobacterium-mediated transformation for gene silencing of developmentally regulated genes, like HvCKX2, was proven. Otherwise low productivity and disturbances in plant development of biolistic-silenced lines documented the unsuitability of the method. The possible reasons are discussed.
doi:10.1186/1471-2229-12-206
PMCID: PMC3541248  PMID: 23134638
RNAi silencing; HvCKX2; Barley; Genetic transformation; Agrobacterium; Microprojectile bombardment
11.  A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase 
PLoS ONE  2014;9(3):e90877.
We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM−1cm−2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.
doi:10.1371/journal.pone.0090877
PMCID: PMC3942484  PMID: 24595403
12.  Co-ordinate regulation of cytokinin gene family members during flag leaf and reproductive development in wheat 
BMC Plant Biology  2012;12:78.
Background
As the global population continues to expand, increasing yield in bread wheat is of critical importance as 20% of the world’s food supply is sourced from this cereal. Several recent studies of the molecular basis of grain yield indicate that the cytokinins are a key factor in determining grain yield. In this study, cytokinin gene family members in bread wheat were isolated from four multigene families which regulate cytokinin synthesis and metabolism, the isopentenyl transferases (IPT), cytokinin oxidases (CKX), zeatin O-glucosyltransferases (ZOG), and β-glucosidases (GLU). As bread wheat is hexaploid, each gene family is also likely to be represented on the A, B and D genomes. By using a novel strategy of qRT-PCR with locus-specific primers shared among the three homoeologues of each family member, detailed expression profiles are provided of family members of these multigene families expressed during leaf, spike and seed development.
Results
The expression patterns of individual members of the IPT, CKX, ZOG, and GLU multigene families in wheat are shown to be tissue- and developmentally-specific. For instance, TaIPT2 and TaCKX1 were the most highly expressed family members during early seed development, with relative expression levels of up to 90- and 900-fold higher, respectively, than those in the lowest expressed samples. The expression of two cis-ZOG genes was sharply increased in older leaves, while an extremely high mRNA level of TaGLU1-1 was detected in young leaves.
Conclusions
Key genes with tissue- and developmentally-specific expression have been identified which would be prime targets for genetic manipulation towards yield improvement in bread wheat breeding programmes, utilising TILLING and MAS strategies.
doi:10.1186/1471-2229-12-78
PMCID: PMC3410795  PMID: 22672647
13.  Cytokinins 
Cytokinins are N6 substituted adenine derivatives that affect many aspects of plant growth and development, including cell division, shoot initiation and growth, leaf senescence, apical dominance, sink/source relationships, nutrient uptake, phyllotaxis, and vascular, gametophyte, and embryonic development, as well as the response to biotic and abiotic factors. Molecular genetic studies in Arabidopsis have helped elucidate the mechanisms underlying the function of this phytohormone in plants. Here, we review our current understanding of cytokinin biosynthesis and signaling in Arabidopsis, the latter of which is similar to bacterial two-component phosphorelays. We discuss the perception of cytokinin by the ER-localized histidine kinase receptors, the role of the AHPs in mediating the transfer of the phosphoryl group from the receptors to the response regulators (ARRs), and finally the role of the large ARR family in cytokinin function. The identification and genetic manipulation of the genes involved in cytokinin metabolism and signaling have helped illuminate the roles of cytokinins in Arabidopsis. We discuss these diverse roles, and how other signaling pathways influence cytokinin levels and sensitivity though modulation of the expression of cytokinin signaling and metabolic genes.
doi:10.1199/tab.0168
PMCID: PMC3894907  PMID: 24465173
14.  tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.†‡  
Journal of Bacteriology  2002;184(7):1832-1842.
Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.
doi:10.1128/JB.184.7.1832-1842.2002
PMCID: PMC134930  PMID: 11889088
15.  Differential spatial expression of A- and B-type CDKs, and distribution of auxins and cytokinins in the open transverse root apical meristem of Cucurbita maxima 
Annals of Botany  2010;107(7):1223-1234.
Background and Aims
Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima.
Methods
Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization.
Key Results
The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM.
Conclusions
Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.
doi:10.1093/aob/mcq127
PMCID: PMC3091794  PMID: 20601387
Auxin; cytokinins; CDKs; Cucurbita maxima; root apical meristems
16.  Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop 
Annals of Botany  2013;111(4):551-562.
Background and Aims
Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation.
Methods
Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed.
Key Results
The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein.
Conclusions
Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.
doi:10.1093/aob/mct012
PMCID: PMC3605948  PMID: 23388879
Apoplast; carboxyfluorescein; floret; glucose; membrane transport; phloem; reflection coefficient; sucrose; symplast; turgor pressure; Zea mays
17.  A natural antisense transcript of the Petunia hybrida Sho gene suggests a role for an antisense mechanism in cytokinin regulation 
The Plant Journal   2007;52(6):1131-1139.
The Sho gene from Petunia hybrida encodes an enzyme responsible for the synthesis of plant cytokinins. The 3′ region of the Sho gene contains a promoter in the opposite orientation that produces a partially overlapping antisense transcript. Although Sho expression varies significantly in individual cell types, the sense and antisense transcript levels maintain a stable ratio in most tissue types. In reporter lines for the antisense promoter, we observed a change in antisense promoter activity in newly formed tissue that had been induced by prolonged culture on cytokinins or following decapitation. We interpret these data as a reflection of tissue-specific threshold levels for activation of the antisense transcript. In all tissue types tested, we detect a pool of antisense RNA of approximately 35 nt, which derives from the region where Sho sense and antisense transcripts overlap. We detect a second pool of putative dsRNA breakdown products of approximately 24 nt in all tissues tested, except roots, which are the main source of cytokinin synthesis. Our data suggest that antisense transcription can be activated in a tissue-specific manner to adjust local cytokinin synthesis via degradation of Sho dsRNA. We therefore propose that, in addition to cytokinin transport and inactivation, regulation of local cytokinin synthesis via antisense transcription represents yet another device for the complex control of local cytokinin levels in plants.
doi:10.1111/j.1365-313X.2007.03309.x
PMCID: PMC2253869  PMID: 17944812
natural antisense transcript; cytokinin; small RNA; RNA turnover; plant hormone; Petunia hybrida
18.  Overexpression of Cytokinin Dehydrogenase Genes in Barley (Hordeum vulgare cv. Golden Promise) Fundamentally Affects Morphology and Fertility 
PLoS ONE  2013;8(11):e79029.
Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
doi:10.1371/journal.pone.0079029
PMCID: PMC3829838  PMID: 24260147
19.  Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation 
Journal of Experimental Botany  2008;59(10):2673-2686.
To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.
doi:10.1093/jxb/ern137
PMCID: PMC2486472  PMID: 18515825
Corn; cytokinin; plant development; tasselseed; Zea mays; zeatin O-glucosyltransferase
20.  Relationship of source and sink in determining kernel composition of maize 
Journal of Experimental Botany  2009;61(2):511-519.
The relative role of the maternal source and the filial sink in controlling the composition of maize (Zea mays L.) kernels is unclear and may be influenced by the genotype and the N supply. The objective of this study was to determine the influence of assimilate supply from the vegetative source and utilization of assimilates by the grain sink on the final composition of maize kernels. Intermated B73×Mo17 recombinant inbred lines (IBM RILs) which displayed contrasting concentrations of endosperm starch were grown in the field with deficient or sufficient N, and the source supply altered by ear truncation (45% reduction) at 15 d after pollination (DAP). The assimilate supply into the kernels was determined at 19 DAP using the agar trap technique, and the final kernel composition was measured. The influence of N supply and kernel ear position on final kernel composition was also determined for a commercial hybrid. Concentrations of kernel protein and starch could be altered by genotype or the N supply, but remained fairly constant along the length of the ear. Ear truncation also produced a range of variation in endosperm starch and protein concentrations. The C/N ratio of the assimilate supply at 19 DAP was directly related to the final kernel composition, with an inverse relationship between the concentrations of starch and protein in the mature endosperm. The accumulation of kernel starch and protein in maize is uniform along the ear, yet adaptable within genotypic limits, suggesting that kernel composition is source limited in maize.
doi:10.1093/jxb/erp324
PMCID: PMC2803218  PMID: 19917600
C/N; carbon; grain; maternal; nitrogen; protein; sink; source; starch; transport
21.  AHP6 Inhibits Cytokinin Signaling to Regulate the Orientation of Pericycle Cell Division during Lateral Root Initiation 
PLoS ONE  2013;8(2):e56370.
In Arabidopsis thaliana, lateral roots (LRs) initiate from anticlinal cell divisions of pericycle founder cells. The formation of LR primordia is regulated antagonistically by the phytohormones cytokinin and auxin. It has previously been shown that cytokinin has an inhibitory effect on the patterning events occurring during LR formation. However, the molecular players involved in cytokinin repression are still unknown. In a similar manner to protoxylem formation in Arabidopsis roots, in which AHP6 (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6) acts as a cytokinin inhibitor, we reveal that AHP6 also functions as a cytokinin repressor during early stages of LR development. We show that AHP6 is expressed at different developmental stages during LR formation and is required for the correct orientation of cell divisions at the onset of LR development. Moreover, we demonstrate that AHP6 influences the localization of the auxin efflux carrier PIN1, which is necessary for patterning the LR primordia. In summary, we show that the inhibition of cytokinin signaling through AHP6 is required to establish the correct pattern during LR initiation.
doi:10.1371/journal.pone.0056370
PMCID: PMC3572949  PMID: 23457561
22.  ENOD40 Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant, Masym3, of Melilotus alba Desr. 
Plant Signaling & Behavior  2007;2(1):33-42.
Several nonnodulating, nonmycorrhizal (Nod−Myc−) mutants of Melilotus alba Desr. (white sweetclover) have been described. However, the details of their responses to Sinorhizobium meliloti have not been fully elucidated. We investigated rhizobial entry and colonization using Confocal Scanning Laser Microscopy on the Masym1–5 mutants and isolated an early nodulin (ENOD40) gene from wild-type M. alba. We focused on Masym3, the least responsive of the mutants to S. meliloti and VA-fungi, to determine its response to cytokinin. Cytokinin appears to be a downstream signal in the nodule developmental pathway based not only on our previous observations whereby Nod−Myc− alfalfa roots treated with cytokinin accumulated several ENOD gene transcripts, but also on recent reports showing the importance of cytokinin receptors for nodulation. Here we show that applying 10−6 M 6-benzylaminopurine to uninoculated Masym3 roots elicited ENOD40 transcript accumulation. In addition, Masym3 root hairs inoculated with either wild-type S. meliloti or Nod− S. meliloti expressing the trans-zeatin synthase gene of Agrobacterium tumefaciens exhibited tip swelling, suggesting that cytokinin mediated this response. However, Masym3 root hair tips swelled following inoculation with Nod− S. meliloti or after mock-inoculation, a response resembling the phenotype of root hairs, after handling, of the Medicago truncatula mutant, dmi2. Mtdmi2 is Nod−Myc− due to a defect in a gene encoding a Nodule Receptor Kinase (NORK). Like Mtdmi2, the root hair swelling response appears in part to be mediated by touch because Masym3 root hairs not contacted by either bacteria or drops of water or buffer remain elongated and do not exhibit tip swelling.
PMCID: PMC2633896  PMID: 19516966
cytokinin; early nodulin ENOD40; Melilotus alba; nodulation mutant; root hair
23.  The transcription factor MtSERF1 may function as a nexus between stress and development in somatic embryogenesis in Medicago truncatula 
Plant Signaling & Behavior  2008;3(7):498-500.
In Medicago truncatula high rates of somatic embryo formation can be induced in the Jemalong genotype 2HA by application of the hormones auxin and cytokinin. Biosynthesis of the stress-related hormone ethylene is also necessary for somatic embryogenesis (SE) and is most likely a response to wounding and the presence of auxin in the medium. We have demonstrated that expression of a gene designated Mt SOMATIC EMBRYO RELATED FACTOR 1 (MtSERF1) induced by ethylene, in the presence of auxin plus cytokinin, is essential for SE. The promoter region of this transcription factor, a member of the ERF sub-family of the AP2/ERF super family, contains putative binding sites relating to auxin and cytokinin in addition to ethylene. An additional finding was the presence of WUSCHEL (WUS) binding sites in the MtSERF1 promoter region, which is discussed. Here we also discuss the Medicago data in the context of embryogenesis studies in Arabidopsis and suggest that MtSERF1 has a key developmental role, possibly in conjunction with WUS, in regulating downstream genes required for the initiation of SE.
PMCID: PMC2634442  PMID: 19704498
somatic embryogenesis; MtSERF1; ethylene; auxin; cytokinin; Medicago truncatula
24.  Nitrogen-Dependent Regulation of De Novo Cytokinin Biosynthesis in Rice: The Role of Glutamine Metabolism as an Additional Signal 
Plant and Cell Physiology  2013;54(11):1881-1893.
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.
doi:10.1093/pcp/pct127
PMCID: PMC3814184  PMID: 24058148
Ammonium; Arabidopsis thaliana; Cytokinin; Glutamine; Nitrate; Oryza sativa
25.  Atypical response regulators expressed in the maize endosperm transfer cells link canonical two component systems and seed biology 
BMC Plant Biology  2010;10:84.
Background
Two component systems (TCS) are phosphotransfer-based signal transduction pathways first discovered in bacteria, where they perform most of the sensing tasks. They present a highly modular structure, comprising a receptor with histidine kinase activity and a response regulator which regulates gene expression or interacts with other cell components. A more complex framework is usually found in plants and fungi, in which a third component transfers the phosphate group from the receptor to the response regulator. They play a central role in cytokinin mediated functions in plants, affecting processes such as meristem growth, phyllotaxy, seed development, leaf senescence or tissue differentiation. We have previously reported the expression and cellular localization of a type A response regulator, ZmTCRR-1, in the transfer cells of the maize seed, a tissue critical for seed filling and development, and described its regulation by a tissue specific transcription factor. In this work we investigate the expression and localization of other components of the TCS signalling routes in the maize seed and initiate the characterization of their interactions.
Results
The discovery of a new type A response regulator, ZmTCRR-2, specifically expressed in the transfer cells and controlled by a tissue specific transcription factor suggests a previously unknown role for TCS in the biology of transfer cells. We have characterized other canonical TCS molecules, including 6 histidine kinases and 3 phosphotransfer proteins, potentially involved in the atypical transduction pathway defined by ZmTCRR-1 and 2. We have identified potential upstream interactors for both proteins and shown that they both move into the developing endosperm. Furthermore, ZmTCRR-1 expression in an heterologous system (Arabidopsis thaliana) is directed to xylem parenchyma cells, probably involved in transport processes, one of the major roles attributed to the transfer cell layer.
Conclusions
Our data prove the expression of the effector elements of a TCS route operating in the transfer cells under developmental control. Its possible role in integrating external signals with seed developmental processes is discussed.
doi:10.1186/1471-2229-10-84
PMCID: PMC3017813  PMID: 20459670

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