Genomic analysis of isopentenyltransferase genes and functional characterization of TaIPT8 indicates positive effects of cytokinins on drought tolerance in wheat

Cytokinins(CKs)function in plant development and during stress responses,but their role in drought tolerance in wheat is unknown.In the present study,24 isopentenyltransferase(IPT)genes,encoding ratelimiting enzymes in CK biosynthesis were identified in the wheat genome.The chromosomal locations and structures of the genes,protein properties,and phylogenetic relationships were characterized.ATP/ADP TaIPT genes showed tissue-specific expression.TaIPT2,TaIPT7,and TaIPT8 expression was rapidly induced by 0.5–1 h drought treatments,which decreased to low levels after 2 h drought treatment,as did most other TaIPT genes.TaIPT8-5a/5b/5d triple mutants showed decreased levels of tZtype CK under normal and drought conditions and reduced drought tolerance,which,however,did not manifest as phenotype alterations.By contrast,transgenic wheat plants with drought-induced TaIPT8showed increased drought tolerance.Our study provides a foundation for further investigation of TaIPT genes and novel insights into the role of CKs in the drought response of wheat.

Review: Plant Growth Hormone Cytokinins Control the Crop Seed Yield

This review chronicles the development of the cytokinin research during the last 30 years. Cytokinin and auxin are the two major plant growth hormones that control virtually all aspects of growth and development in higher plants. The pathways for cytokinin biosynthesis and metabolism have been characterized by the identification of isopentenyl pyrophosphate transferase, cytokinin oxidases, cytokinin hydroxylase, zeatin cis-/trans-isomerase, cytokinin phosphoribosyl hydrolases, cytokinin-specific riboside phosphorylase, and others enzymes. Loss-of function mutant phenotypes of cytokinin degradation/activating enzymes indicate the regulation of concentration and spatial distribution of bio-active cytokinin plays a pivotal role in the increase in panicle size, in the numbers of floral organs, and eventually in seed yield. One of the most fundamental questions in the cytokinin field is one concerning the prevalence of cis-zeatin in monocotyledonous crops (rice and maize) and in dicotyledonous legumes (pea, chickpea) and potato/sweet potato. A hypothesis is that cis-zeatin is synthesized by the cis-specific hydroxylation of the terminal methyl group of N6-isopentenyl side chain of N6-isopentenyl adenosine (i6Ado) or of their mono-, di-, or tri-phosphates catalyzed by the cis-specific hydroxylase. A second potential pathway is the isomerization of trans-zeatin to cis-zeatin by zeatin cis-/trans-isomerase. A second fundamental question to be addressed is the physiological role of cis-zeatin. Some have argued for a special function of cis-zeatin to account for the prevalence of the cis-zeatin in the plant kingdom from algae to higher plants.

Effect of Macronutrients, Cytokinins and Auxins, on <i>in Vitro</i>Organogenesis of <i>Thymus vulgaris</i>L.

The present study reports an efficient protocol for in vitro propagation of Thymus vulgaris L., an aromatic and medicinal plant in Morocco. Initially, we performed in vitro multiplication of Thymus vulgaris explants existing in the laboratory and obtained from micropropagation by shoot tip culture. Afterwards, we have evaluated the effect of six macronutrients. After that, seven cytokinins (Kin, BAP, 2iP, DPU, Adenine, Zeatine and TDZ) in three different concentrations (0.46, 0.93, 2.32 μM) have been evaluated to optimize cultures multiplication and elongation. Moreover, the effect of three auxins (IAA, IBA and NAA) at 0.57 μM, combined to 4 cytokinins (Kin, BAP, DPU and Ad.) at 0.46 μM, on shoot rooting has been studied. Thereby, MS medium has been proved the most favorable for plantlets growing. Also, we found that the addition of certain cytokinins, specifically 0.46 Kin, 0.46 and 0.93 BAP, 0.46 2iP, 0.46 DPU, 0.46 Ad. and 0.46 Zeat., ensures better multiplication and growth of vitroplants. In addition, multiplication and rooting of cultures were well optimized after addition 0.46 Kin + 0.57 IAA or NAA, 0.46 DPU + 0.57 IBA and 0.46 Ad. + 0.57 IBA combinations to the culture medium. Lastly, plantlets with roots were successfully acclimatized to ex-vitro conditions and these latter served as a source to establish in vitro culture again.

EXOGENOUS Ca^(2+) INHIBITS SENESCENCE-RETARDING EFFECT OF CYTOKININS IN DETACHED RICE LEAVES

Cytokinins and Ca 2+ singly retarded senescence of detached rice leaves.When Ca 2+ was applied together with cytokinins,the effectiveness of cytokinins was significantly reduced.Ca 2+ and cytokinins did not stimulate ethylene production synergistically,ruling out the possibility that ethylene was involved in the inhibition of cytokinin induced senescence retarding effect by Ca 2+ .The experiment with specific compounds known to increase (Ca ionophore A23187),or decrease (EGTA,LaCl 3,Verapamil,chlorpromazine) cytosolic Ca 2+ level indicated that the elevated cytosolic Ca 2+ retards senescence.

Effect of Cytokinins on the Micropropagation of Carob(Ceratonia siliqua L.)through Shoot Tip Culture

In order to determine the most suitable cytokinin for the micropropagation of carob (Ceratonia siliqua L.), the effect of four cytokinins: BAP, zeatin, kinetin and 2-iP, was tested on explants derived from young seedlings of seven days. Organogenesis is better in the presence of BAP (0.5 mg/l), while buds growth is favored by zeatin (0.5 mg/l). The combination of the most suitable cytokinin (BAP at 0.5 mg/l) with IBA improves the caulogenesis when the concentration of this latter is low (0.1 mg/l);high concentrations of IBA have an inhibitory effect on elongation and neoformation of shoots and leaves. The multiplication and growth of shoots are more favorable on WPM medium in the presence of BAP (0.5 and 1 mg/l) alone or combined with 0.5 mg/l with GA3, while rooting is mainly favored by IBA, especially at 2 mg/l.

Polyamine Alterations in Isolated Zucchini Cotyledons Grown in Presence of Cytokinins and Cu²⁺

Polyamines are small nitrogen-containing organic molecules, which are widely distributed in plants. They are involved in the regulation of normal plant growth and developmental processes. In this study we examined the role of polyamines on the growth of Cucurbita pepo L.(zucchini) cotyledons incubated on solutions of different types of cytokinins (BA—N6-benzylaminopurine or 4PU-30—N1-(2-chloro-4-pyridyl)-N2-phenylurea) and copper in excess. We found that endogenous polyamines, and mainly the conjugated fraction, are involved in the cell division processes of isolated zucchini cotyledons and their changes are related to the specific action of the used growth regulating factors.

Seasonal changes of endogenous ABA and cytokinins in environmental adaptation of different ecotypes of reed plants

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EFFECT OF CYTOKININS AND AUXINS ON THE GROWTH OF FREE-LIVING CONCHOCELIS OF PORPHYRA YEZOENSIS

1AA 3-Indolylacetic acid, NAA a-Naphthylacetic acid and cytokinins in PESI culture medium were used in a study on the effects of plant hormones on the growth of free-living conchocelis of Porphyra yezoensis which showed that its growth in medium with cytokinins, 1AA and NAA was more rapid than that in medium with non-phytohormones; that the optimal concentrations for promoting growth were 10μg/L for IAA and ZA (Zeatin), and 0.1 μg/L for BA 6-Benzyl amino purine and KIN 6-Furfurylamino- purine. Mix use of NAA, 1AA and cytokinins, NAA/ZA 1-1000/1μg/L, NAA/BA 10/1-1000 μg/L, NAA/KIN 1/1-1000 μg/L promoted growth. 1AA/ZA 0.1-1/0.1-1μg/L; 1AA/BA 0.1-1/0.1-10 μg/L IAA/KIN 1/0.1-1000μg/L also promoted growth.

Spatially distributed cytokinins:Metabolism,signaling,and transport

Cytokinins are mobile phytohormones that regulate plant growth,development,and environmental adaptability.The major cytokinin species include isopentenyl adenine(iP),trans-zeatin(tZ),cis-zeatin(cZ),and dihydrozeatin(DZ).The spatial distributions of different cytokinin species in different organelles,cells,tissues,and organs are primarily shaped by biosynthesis via isopentenyltransferases(IPT),cytochrome P450 monooxygenase,and 50-ribonucleotide phosphohydrolase and by conjugation or catabolism via glycosyltransferase or cytokinin oxidase/dehydrogenase.Cytokinins bind to histidine receptor kinases in the endoplasmic reticulum or plasma membrane and relay signals to response regulators in the nucleus via shuttle proteins known as histidine phosphotransfer proteins.The movements of cytokinins from sites of biosynthesis to sites of signal perception usually require long-distance,intercellular,and intracellular transport.In the past decade,ATP-binding cassette(ABC)transporters,purine permeases(PUP),AZA-GUANINE RESISTANT(AZG)transporters,equilibrative nucleoside transporters(ENT),and Sugars Will Eventually Be Exported transporters(SWEET)have been characterized as involved in cytokinin transport processes.This review begins by introducing the spatial distributions of various cytokinins and the subcellular localizations of the proteins involved in their metabolism and signaling.Highlights focus on an inventory of the characterized transporters involved in cytokinin compartmentalization,including long-distance,intercellular,and intracellular transport,and the regulation of the spatial distributions of cytokinins by environmental cues.Future directions for cytokinin research are also discussed.

Arabidopsis ABCG14 forms a homodimeric transporter for multiple cytokinins and mediates long-distance transport of isopentenyladeninetype cytokinins

Cytokinins(CKs),primarily trans-zeatin(tZ)and isopentenyladenine(iP)types,play critical roles in plant growth,development,and various stress responses.Long-distance transport of tZ-type CKs meidated by Arabidopsis ATP-binding cassette transporter subfamily G14(AtABCG14)has been well studied;however,less is known about the biochemical properties of AtABCG14 and its transporter activity toward iP-type CKs.Here we reveal the biochemical properties of AtABCG14 and provide evidence that it is also required for long-distance transport of iP-type CKs.AtABCG14 formed homodimers in human(Homo sapiens)HEK293T,tobacco(Nicotiana tabacum),and Arabidopsis cells.Transporter activity assays of AtABCG14 in Arabidopsis,tobacco,and yeast(Saccharomyces cerevisiae)showed that AtABCG14 may directly transport multiple CKs,including iP-and tZ-type species.AtABCG14 expression was induced by iP in a tZ-type CKdeficient double mutant(cypDM)of CYP735A1 and CYP735A2.The atabcg14 cypDM triple mutant exhibited stronger CK-deficiency phenotypes than cypDM.Hormone profiling,reciprocal grafting,and 2H6-iP isotope tracer experiments showed that root-to-shoot and shoot-to-root long-distance transport of iP-type CKs were suppressed in atabcg14 cypDM and atabcg14.These results suggest that AtABCG14 participates in three steps of the circular long-distance transport of iP-type CKs:xylem loading in the root for shootward transport,phloem unloading in the shoot for shoot distribution,and phloemunloading in the root for root distribution.We found that AtABCG14 displays transporter activity toward multiple CK species and revealed its versatile roles in circular long-distance transport of iP-type CKs.These findings provide newinsights into the transport mechanisms of CKs and other plant hormones.

PbGIF1 promoting cell-proliferation in pear fruit is transcriptionally activated by Pb RR1

As a cell proliferation regulator involved in wide biological processes in plants,GRF-INTERACTING FACTOR(GIF)controls different tissues development.However,whether GIF participates in fruit development remains unclear.According to transcriptome data,we identified PbGIF1was highly expressed during fruit development in cytokinins induced parthenocarpy pear.In the present study,the biofunction of PbGIF1 was initially verified.Overexpression of PbGIF1 promoted fruit size of transgenic tomato.The size of flesh fruit was not affected by cell expansion but the cell proliferation was promoted by overexpressing Pb GIF1.The accelerated cell proliferation process was also observed in PbGIF1-overexpressed transgenic pear fruit calli.The transcriptional regulation of cytokinins on PbGIF1 was further confirmed by exogenous CPPU treatments in pear fruitlets.To investigate the underlying mechanism,the cytokinins-responded factor,PbRR1,was further focused on.The results of Yeast-one-hybrid assay suggested that PbRR1 can bind to the promoter sequence of PbGIF1.The transcriptional activation of PbRR1 on PbGIF1 was also confirmed by Dual-Luciferase assays.Taken together,the results showed that cytokinins control pear fruit development via the transcriptional activation of PbGIF1 by PbRR1.

Metabolism and Long-distance Translocationof Cytokinins

During plant development, distantly-located organs must communicate in order to adapt morphological and physiological features in response to environmental inputs. Among the recognized signaling molecules, a class of phytohormones known as the cytokinins functions as both local and long-distance regulatory signals for the coordination of plant development. This cytokinin-dependent communication system consists of orchestrated regulation of the metabolism, translocation, and signal transduction of this phytohormone class. Here, to gain insight into this elaborate signaling system, we summarize current models of biosynthesis, transmembrane transport, and long-distance translocation of cytokinins in higher plants.

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maize miniature1 Seed Mutant and Its Wild Type

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplicaUon in developing maize caryopsis of a cell wall invertase-deficient miniature1 （mn1） seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mnl compared with mnl, resulting in a significantly higher cell number in the Mnl endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 （INCW2） at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

Dynamics and origin of cytokinins involved in plant manipulation by a leaf-mining insect

Several herbivorous insects and plant-associated microorganisms control the phytohormonal balance, thus enabling them to successfully exploit the plant by inhibiting plant defenses and withdrawing plant resources for their own benefit. The leaf-mining moth Phyllonorycter blancardella modifies the cytokinin （CK） profile of mined leaf-tissues, and the insect symbiotic bacteria Wolbachia is involved in the plant manipulation to the benefit of the insect host. To gain a deeper understanding into the possible origin and dynamics of CKs, we conducted an extensive characterization of CKs in larvae and in infected apple leaves. Our results show the enhanced CK levels in mines, both on green and yellow leaves, allowing insects to control their nutritional supply under fluctuating environmental conditions. The spatial distribution of CKs within the mined leaves shows that hormone manipulation is strictly limited to the mine suggesting the absence of CK translocation from distant leaf areas toward the insect feeding site. Mass spectrometry analyses reveal that major CK types accumulating in mines and larvae are similar to what is observed for most gall-inducers, suggesting that strategies underlying the plant manipulation may be shared between herbivorous insects with distinct life histories. Results further show that CKs are detected in the highest levels in larvae, reinforcing our hypothesis that CKs accumulating in the mines originate from the insect itself. Presence of bacteria- specific methylthio-CKs is consistent with previous results suggesting that insect bacterial symbionts contribute to the observed phenotype. Our study provides key findings toward the understanding of molecular mechanisms underlying this intricate plant-insect-microbe interaction.

Improving dryland maize productivity and water efficiency with heterotrophic ammonia-oxidizing bacteria via nitrification and cytokinin activity

A two-year field experiment conducted under dryland conditions in semi-humid and drought-prone regions of China aimed to assess the effect of ammonia-oxidizing bacterial on maize water use efficiency and yield.A heterotrophic ammonia-oxidizing bacteria(HAOB)strain S2_8_1 was used.Six treatments were applied:(1)no irrigation+HAOB strain(DI),(2)no irrigation+blank culture medium(DM),(3)no irrigation control(DCK),(4)irrigation+HAOB(WI),(5)irrigation+blank culture medium(WM),and(6)irrigation control(WCK).Results revealed that HAOB treatment increased maize growth,yield,and water use efficiency over controls,regardless of whether the year was wet or dry.This improvement was attributed to the accelerated nitrification in the rhizosphere soil due to HAOB inoculation,which subsequently led to increased levels of leaf cytokinins.Overall,these findings suggest that HAOB inoculation holds promise as a strategy to boost water use efficiency and maize productivity in dryland agriculture.

Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides （LCOs）. Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accu- mulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizo- bium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and iso- pentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase （CCaMK）, a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein21 Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

ETR1 Integrates Response to Ethylene and Cytokinins into a Single Multistep Phosphorelay Pathway to Control Root Growth

Cytokinins and ethylene control plant development via sensors from the histidine kinase(HK)family.However,downstream signaling pathways for the key phytohormones are distinct.Here we report that not only cytokinin but also ethylene is able to control root apical meristem(RAM)size through activation of the multistep phosphorelay(MSP)pathway.We found that both cytokinin and ethylene-dependent RAM shortening requires ethylene binding to ETR1 and the HK activity of ETR1.The receiver domain of ETR1 interacts with MSP signaling intermediates acting downstream of cytokinin receptors,further substantiating the role of ETR1 in MSP signaling.We revealed that both cytokinin and ethylene induce the MSP in similar and distinct cell types with ETR1-mediated ethylene signaling controlling MSP output specifically in the root transition zone.We identified members of the MSP pathway specific and common to both hormones and showed that ETR1-regulated ARR3 controls RAM size.ETR1-mediated MSP spatially differs from canonical CTR1/EIN2/EIN3 ethylene signaling and is independent of EIN2,indicating that both pathways can be spatially and functionally separated.Furthermore,we demonstrated that canonical ethylene signaling controls MSP responsiveness to cytokinin specifically in the root transition zone,presumably via regulation of ARR10,one of the positive regulators of MSP signaling in Arabidopsis.

The leaf senescence-promoting transcription factor AtNAP activates its direct target gene CYTOKININ OXIDASE 3 to facilitate senescence processes by degrading cytokinins

Cytokinins(CKs)are a class of adenine-derived plant hormones that plays pervasive roles in plant growth and development including cell division,morphogenesis,lateral bud outgrowth,leaf expansion and senescence.CKs as a“fountain of youth”prolongs leaf longevity by inhibiting leaf senescence,and therefore must be catabolized for senescence to occur.AtNAP,a senescence-specific transcription factor has a key role in promoting leaf senescence.The role of AtNAP in regulating CK catabolism is unknown.Here we report the identification and characterization of AtNAP-AtCKX3(cytokinin oxidase 3)module by which CKs are catabolized during leaf senescence in Arabidopsis.Like AtNAP,AtCKX3 is highly upregulated during leaf senescence.When AtNAP is chemically induced AtCKX3 is co-induced;and when AtNAP is knocked out,the expression of AtCKX3 is abolished.AtNAP physically binds to the cis element of the AtCKX3 promoter to direct its expression as revealed by yeast one-hybrid assays and in planta experiments.Leaves of the atckx3 knockout lines have higher CK concentrations and a delayed senescence phenotype compared with those of WT.In contrast,leaves with inducible expression of AtCKX3 have lower CK concentrations and exhibit a precocious senescence phenotype compared with WT.This research reveals that AtNAP transcription factor˗AtCKX3 module regulates leaf senescence by connecting two antagonist plant hormones abscisic acid and CKs.

Involvement of cytokinins in STOP1-mediated resistance to proton toxicity

STOP1(sensitive to proton rhizotoxicity1)is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH(i.e.,proton)stresses in Arabidopsis.However,the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear.Here,we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities.Interestingly,the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type.Significantly,low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses.Moreover,lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins.Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root.Furthermore,the expression of critical genes involved in cytokinin biosynthesis,biodegradation,and signaling is altered in the stop1 mutant in response to low-pH stresses.In conclusion,our results reveal a complex network of resistance to low-pH stresses,which involves coordinated actions of STOP1,cytokinins,and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.

Grain yield,nitrogen use efficiency and physiological performance of indica/japonica hybrid rice in response to various nitrogen rates

Utilizing the heterosis of indica/japonica hybrid rice(IJHR)is an effective way to further increase rice grain yield.Rational application of nitrogen(N)fertilizer plays a very important role in using the heterosis of IJHR to achieve its great yield potential.However,the responses of the grain yield and N utilization of IJHR to N application rates and the underlying physiological mechanism remain elusive.The purpose of this study was to clarify these issues.Three rice cultivars currently used in rice production,an IJHR cultivar Yongyou 2640(YY2640),a japonica cultivar Lianjing 7(LJ-7)and an indica cultivar Yangdao 6(YD-6),were grown in the field with six N rates(0,100,200,300,400,and 500 kg ha^(-1))in 2018 and 2019.The results showed that with the increase in N application rates,the grain yield of each test cultivar increased at first and then decreased,and the highest grain yield was at the N rate of 400 kg ha^(-1)for YY2640,with a grain yield of 13.4 t ha^(-1),and at 300 kg ha^(-1)for LJ-7 and YD-6,with grain yields of 9.4–10.6 t ha^(-1).The grain yield and N use efficiency(NUE)of YY2640 were higher than those of LJ-7 or YD-6 at the same N rate,especially at the higher N rates.When compared with LJ-7 or YD-6,YY2640 exhibited better physiological traits,including greater root oxidation activity and leaf photosynthetic rate,higher cytokinin content in the roots and leaves,and more remobilization of assimilates from the stem to the grain during grain filling.The results suggest that IJHR could attain both higher grain yield and higher NUE than inbred rice at either low or high N application rates.Improved shoot and root traits of the IJHR contribute to its higher grain yield and NUE,and a higher content of cytokinins in the IJHR plants plays a vital role in their responses to N application rates and also benefits other physiological processes.