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.

Physiological and molecular mechanisms of cytokinin involvement in nitrate-mediated adventitious root formation in apples

Potassium nitrate(KNO_(3))promotes adventitious root(AR)formation in apple stem cuttings.However,evidence for the possible involvement of cytokinin(CK)in KNO_(3)-mediated AR formation in apples is still lacking.In this study,we cultured GL-3 apple microshoots in different treatment combinations.While the T1(KNO_(3)9.4 mmol L^(-1)+6-benzyl adenine(6-BA)2.22μmol L^(-1))and T3(6-BA 2.22μmol L^(-1))treatments completely inhibited AR formation,the control,T2(KNO_(3)9.4 mmol L^(-1)),and T4(KNO_(3)9.4 mmol L^(-1)+lovastatin(Lov)1.24μmol L^(-1))treatments developed ARs.However,T4-treated microshoots developed fewer and shorter ARs,indicating that optimum CK synthesis is needed for normal AR growth.This also suggests that these fewer and shorter ARs developed because of the presence of KNO_(3) in the same medium.The anatomy of the stem basal part indicated that the inhibition of CK biosynthesis delayed AR primordia formation.The endogenous levels of indole-3-acetic acid(IAA)and zeatin riboside(ZR)were higher in T2-treated microshoots,while the abscisic acid(ABA),gibberellic acid 3(GA_(3)),and brassinosteroid(BR)levels were higher in T4-treated microshoots.The expression levels of MdNRT1.1and MdNRT2.1 were higher in T2-treated microshoots at 3 and 8 days,while MdRR2 and MdCKX5 were higher at 8 and 16 days,respectively.Furthermore,higher IAA levels increased MdWOX11 expression,which in turn increased MdLBD16 and MdLBD29 expression in response to T2.The combined expression of these genes stimulated adventitious rooting by upregulating cell cycle-related genes(MdCYCD1;1 and MdCYCD3;1)in response to T2 treatment.This study shows that specific genes and hormonal pathways contribute to KNO_(3)-CK-mediated adventitious rooting in apples.

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.

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.

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.

Genome-wide comparative analysis of type-A Arabidopsis response regulator genes by overexpression studies reveals their diverse roles and regulatory mechanisms in cytokinin signaling

Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the receptors, through histidine phosphotransfer proteins, to the downstream response regulators （ARRs）. Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of eytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and development is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARR5-ARR6 pairs. We found that the accumulation of a subset of ARR proteins （ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15） is increased by MG132, a specific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. Moreover, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARR15, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.

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

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The BUD2 mutation affects plant architecture through altering cytokinin and auxin responses in Arabidopsis

The ratio of auxin and cytokinin plays a crucial role in regulating aerial architecture by promoting or repressing axillary bud outgrowth. We have previously identified an Arabidopsis mutant bud2 that displays altered root and shoot architecture, which results from the loss-of-function of S-adenosylmethionine decarboxylase 4 （SAMDC4）. In this study, we demonstrate that BUD2 could be induced by auxin, and the induction is dependent on auxin signaling. The mutation of BUD2 results in hyposensitivity to auxin and hypersensitivity to cytokinin, which is confirmed by callus induction assays. Our study suggests that polyamines may play their roles in regulating the plant architecture through affecting the homeostasis of cytokinins and sensitivities to auxin and cytokinin.

TaARR1, a cytokinin response regulator gene in Triticum aestivum, is essential in plant N starvation tolerance via regulating the N acquisition and N assimilation

Plant N starvation response is closely associated with the N signaling components that involve transduction of the low-N cues. In this study, we functionally characterized Ta ARR1, a cytokinin(CK) response regulator gene in Triticum aestivum, in mediating the N starvation adaptation in plants. Ta ARR1 harbors two conserved domains specified by plant ARR family members;subcellular localization analysis indicated its target onto nucleus after endoplasmic reticulum assortment. Ta ARR1 displayed modified expression upon the N starvation stressor, showing upregulated expression in roots and leaves over a 27-h N starvation treatment and whose induced transcripts were gradually recovered along with progression of the N recovery treatment. The tobacco lines overexpressing Ta ARR1 displayed improved low-N stress tolerance, displaying enlarged phenotype, increased biomass and N accumulation, and enhanced glutamine synthetase(GS) activities compared with wild type(WT) following the N starvation treatment. Expression analysis on genes encoding the nitrate transporter(NRT) and GS proteins in Nicotiana tabacum revealed that Nt NRT2.2 and Nt GS3 are upregulated in expression in the N-deprived transgenic lines, whose expression patterns were contrasted to other above family genes that were unaltered on transcripts between the transgenic lines and WT. Transgene analysis validated the function of Nt NRT2.2 and Nt GS3 in regulating N accumulation, GS activity, growth traits, and N use efficiency in plants. These results suggested the internal connection between the Ta ARR1-mediated N starvation tolerance and the modified transcription of distinct N acquisitionand assimilation-associated genes. Our investigation together indicates that Ta ARR1 is essential in plant N starvation adaptation due to the gene function in transcriptionally regulating distinct NRT and GS genes that affect plant N uptake and assimilation under the N starvation condition.

Interactions between cytokinin and nitrogen contribute to grain mass in wheat cultivars by regulating the flag leaf senescence process

Premature senescence after anthesis reduces crop yields.Delaying leaf senescence could maintain photosynthetic activity for a longer period and lead to a higher photosynthetic rate.Recent studies have provided some insights into the interaction between cytokinin and nitrogen(N)in the regulation of plant development.In the present study,foliar application of exogenous 6-benzylaminopurine(6-BA)and lovastatin,an inhibitor of cytokinin synthesis,was combined with three N rates[0 kg ha^(-1)(low nitrogen,LN),240 kg ha^(-1)(normal nitrogen,NN),and 360 kg ha^(-1)(high nitrogen,HN)]in two wheat cultivars,Wennong 6(with a staygreen phenotype)and Jimai 20(with a non-staygreen phenotype).Flag leaf senescence was assessed using a Gompertz growth curve.Grain mass,dry matter accumulation and distribution,total N of flag leaf,and concentrations of zeatin riboside(ZR)and abscisic acid(ABA)were also used to evaluate the functional characteristics of flag leaves.Grain mass was negatively correlated with initial senescence rate(r_0)and duration of rapid chlorophyll loss(Chl_(loss)),whereas it was positively correlated with maximum senescence rate(r_(max)),average senescence rate(r_(aver)),persistence phase(Chl_(per)),total duration of flag leaf(Chl_(total))and inflection point cumulative temperature(M).Compared to Jimai 20,Wennong 6 had larger r_(aver),Chl_(per),and Chl_(total).The concentration of ZR was highest under the 6-BA×NN treatment,followed by the 6-BA×HN and 6-BA×LN treatments.However,the concentration of ABA showed the opposite trend.It was concluded that the staygreen phenotype Wennong 6 was associated with greater grain mass and altered cytokinin metabolism and could be classified as a functional staygreen type.Foliar application of 6-BA interacting with N at the NN level(240 kg ha^(-1))may be a beneficial strategy for improving grain yield of wheat by regulating endogenous hormones and the flag leaf senescence process.Increasing endogenous cytokinin promoted the transport of dry matter to grain.

Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L.(Scrophulariaceae)-an ethnomedicinal herb

Objective:To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis(S.dulcis) L.Methods:Explants were inoculated on MS basal medium supplemented with kinelin and 6-benzylaminopurine for shoot bud induction.To enhance the shoot induction,various auxins like 3-indoleacetic acid or 3-indolebutyric acid or a-naphthylacetic acid were tested along with 2.32 M KI and 4.44 μ M BAP.The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA,IBA or NAA.After roots were developed,the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%-80%humidity under 16 h photoperiod.After acclimatization,the plantlets were transferred to the garden and survival percentage was calculated.Data were statistically analyzed and means were compared using Duncan's multiple range test(P<0.05).Results:An in vitro method was developed to induce high frequency shoots regeneration from stem,mature leaf and young leaf explants of S.dulcis.Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins(2.32 μ M KI and 4.44 μ M BAP) 2.85 μ M IAA,10%CM and 1 483.79 μM adenine sulfate.A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP.Conclusions: Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S.dulcis.

Genome-wide identification and analysis of cytokinin dehydrogenase/oxidase(CKX) family genes in Brassica oleracea L.reveals their involvement in response to Plasmodiophora brassicae infections

Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens.However,a comprehensive analysis of cytokinin dehydrogenase/oxidase(CKX)family genes in cabbage has not been reported.In this study,a total of 36 CKX genes were identified using a genome-wide search method.Phylogenetic analysis classified these genes into three groups.They were distributed unevenly across nine chromosomes in B.oleracea,and 15 of them did not contain any introns.The results of colinearity analysis showed that 36 CKX gene in Arabidopsis was present in several copies in the Brassica oleracea genome.An analysis of cisacting elements indicated that all genes possessed at least one stress or hormone responsive cis-acting element.A heatmap of CKX gene expression showed the patterns of expression of these genes in various tissues and organs.Three genes(Bol028363,Bol031036 and Bol018140)were relatively highly expressed in all of the investigated tissues under normal conditions,showing the expression profile of housekeeping genes.Generally,the expression patterns of CKX genes in Jingfeng 1 and Xiangan 336 were quite different under the same treatment.Notably,three genes(Bol020547,Bol028392 and Bol045724)were significantly down-regulated and up-regulated in the susceptible and resistant material,respectively,after inoculation,which may indicate their crucial roles in resistance to clubroot disease.The results provide insights for better understanding the roles of CKX genes in the B.oleracea–P.brassicae interaction.

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.

Quantitative Studies on Structure－Activity Relationships （QSAR） of Cytokinin－Active Phenyl Urea Derivatives（PUD）

he quantitative studies on structure and cytokinin activity of the cucumber cotyledon expansion reiationshps of 35 urea compounds were conducted using pl1ysico-chemical parameters of the substituents and regression analysis,The position-specific hydrophobic,steric and electronic effects of substituents are important to the activity.A certain overlap of the optimum conformation of the ureido structure over Kinetin was observed.The coplanarity is discussed.

IDENTIFICATION OF CYTOKININ IN A GREEN ALGAE EXTRACT

Jsopentenyladenosine (i6 Ado) was identified, and trans-zeatin (trans-Z) and trans -zeatin riboside (trans-ZR) were detected by high pressure liquid chromatography (HPLC) but not verified with chromatography-mass spectrometry (GC-MS) analysis of cytokinin from the extracts of green algae (Ulva pertusa (Kjellm), Enteromopha compressa and Monostroma sp.). This indicated that the green algae mixture contained cytokinin - like substances.

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.

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.

ARR5 and ARR6 Mediate Tissue Specific Cross-talk between Auxin and Cytokinin in <i>Arabidopsis</i>

Auxin and cytokinin interaction plays an essential role in a wide range of plant growth and developmental processes. The interaction consequence of the two hormones is highly tissue specific. The molecular mechanisms underlying the tissue specificity are largely unknown. Here we show that the cytokinin signaling pathway key components ARR5 and ARR6 respond to auxin with a highly tissue specific and contrasted pattern in Arabidopsis seedlings and calli in the presence of cytokinin. Our results suggest that the two highly homologous but functionally distinct genes, ARR5 and ARR6, play a critical role in mediating tissue specific interaction between auxin and cytokinin.

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.

Foliar Application of Cytokinin Modulates Gas Exchange Features,Water Relation and Biochemical Responses to Improve Growth Performance of Maize under Drought Stress

Improvement of plant performance under drought stress is crucial to sustaining agricultural productivity.The current study investigated the ameliorative effects of foliar-applied kinetin,an adenine-type cytokinin(CK),on growth and gas exchange parameters,water relations and biochemical attributes of maize plants under drought stress.Eighteen-day-old maize plants were subjected to drought by maintaining soil moisture content at 25%field capacity for 8 days followed by foliar application of kinetin at 0,75,150 and 225 mg L^(−1)(CK0,CK75,CK150 and CK225,respectively)to the plants for two-times at the 9-day interval.Results revealed that drought stress markedly reduced stem diameter,dry weight,chlorophyll content,gas exchange parameters and water balance but increased proline,malondialdehyde and soluble sugar contents,electrolyte leakage and senescence in maize leaves.Application of exogenous CK remarkably improved maize performance by modulating growth,gas exchange-and water relation-related parameters in a dose-dependent manner under drought stress.CK225 increased chlorophyll content(by 61.54%),relative water content(by 49.14%),net photosynthesis rate(by 39.94%)and transpiration rate(by 121.36%)and also delayed leaf senescence but decreased internal CO_(2)concentration(by 7.38%),water saturation deficit(by 40.40%)and water uptake capacity(by 42.49%)in both well-watered and droughtstressed plants.Nevertheless,CK application considerably decreased electrolyte leakage,proline,malondialdehyde and soluble sugar levels in drought-stressed maize plants,as also supported by heatmap and cluster analyses.Taken together,exogenous CK at proper concentration(225 mg L^(−1))successfully improved maize performance under drought conditions,thereby suggesting CK application as a useful approach to alleviate drought-induced adverse effects in maize plants,and perhaps in other important crop plants.