New Insights into the Functions of Cytokinins in Plant Development

Recent breakthroughs in cytokinin research have shed new light on the role of cytokinin in plant development. Loss-of-function mutants of a cytokinin receptor reveal a role for the hormone in establishment of the vasculature during embryonic development. Cytokinin controls the number of early cell divisions via a two-component signaling system. Genetically engineered plants that have a reduced cytokinin content demonstrate the regulatory role of the hormone in control of meristem activity and organ growth during postembryonic development, with opposite roles in roots and shoots. There is increasing evidence from work with transgenic plants and mutant analysis that cytokinins do not perform the previously proposed function as a root-derived signal for the regulation of shoot branching. Root-borne cytokinins might serve as a long-range signal controling other processes at distant sites, such as responding to nutritional status, particularly nitrogen availability.     

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytokinin/Auxin Control of Apical Dominance in Ipomoea nil

Although the concept of apical dominance control by the ratioof cytokinin to auxin is not new, recent experimentation withtransgenic plants has given this concept renewed attention.In the present study, it has been demonstrated that cytokinintreatments can partially reverse the inhibitory effect of auxinon lateral bud outgrowth in intact shoots of Ipomoea nil. Althoughless conclusive, this also appeared to occur in buds of isolatednodes. Auxin inhibited lateral bud outgrowth when applied eitherto the top of the stump of the decapitated shoot or directlyto the bud itself. However, the fact that cytokinin promotiveeffects on bud outgrowth are known to occur when cytokinin isapplied directly to the bud suggests different transport tissuesand/or sites of action for the two hormones. Cytokinin antagonistswere shown in some experiments to have a synergistic effectwith benzyladenine on the promotion of bud outgrowth. If theratio of cytokinin to auxin does control apical dominance, thenthe next critical question is how do these hormones interactin this correlative process? The hypothesis that shoot-derivedauxin inhibits lateral bud outgrowth indirectly by depletingcytokinin content in the shoots via inhibition of its productionin the roots was not supported in the present study which demonstratedthat the repressibility of lateral bud outgrowth by auxin treatmentsat various positions on the shoot was not correlated with proximityto the roots but rather with proximity to the buds. Resultsalso suggested that auxin in subtending mature leaves as wellas that in the shoot apex and adjacent small leaves may contributeto the apical dominance of a shoot. (Received September 24, 1996; Accepted March 16, 1997)     

文心兰试管苗丛生芽高效增殖体系的建立

分别采用细胞分裂素6-BA和Ad对文心兰试管苗增殖的作用进行研究,结果表明：在与0．2mg／L的NAA配合使用时,6-BA含量在2．0～4．0mg／L水平下的培养基较适合试管苗的增殖,最大增殖系数可达7．27,苗生长健壮,叶色鲜绿,适合进一步生根移栽;Ad在1．0～6．0mg／L的范围内增殖系数较小,苗生长缓慢、矮小。但有形成丛生苗的倾向;以2．0mg／L的6～BA和1．0mg／L的Ad组合使用,易形成丛生芽苗且苗体相对较小,增殖系数也较高;接种单株小苗较大苗易形成丛生芽苗;接种连体小芽苗,全部能形成丛生芽苗,增殖系数可达10．07,适合于进一步增殖使用。6-BA与Ad配合使用结合接种连体小芽苗可建立高效的丛生芽苗增殖体系,培养基最佳激素组合为MS 6-BA2．0mg／L Ad1．0mg／L NAA0．2mg／L 3％蔗糖 0．4％琼脂粉。     

Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3

Cytokinins control key processes during plant growth and development, and cytokinin receptors CYTOKININ RESPONSE 1/WOODEN LEG/ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/WOL/AHK4), AHK2, and AHK3 have been shown to play a crucial role in this control. The involvement of cytokinins in signaling the status of several nutrients, such as sugar, nitrogen, sulfur, and phosphate (Pi), has also been highlighted, although the full physiological relevance of this role remains unclear. To gain further insights into this aspect of cytokinin action, we characterized a mutant with reduced sensitivity to cytokinin repression of a Pi starvation-responsive reporter gene and show it corresponds to AHK3. As expected, ahk3 displayed reduced responsiveness to cytokinin in callus proliferation and plant growth assays. In addition, ahk3 showed reduced cytokinin repression of several Pi starvation-responsive genes and increased sucrose sensitivity. These effects of the ahk3 mutation were especially evident in combination with the cre1 mutation, indicating partial functional redundancy between these receptors. We examined the effect of these mutations on Pi-starvation responses and found that the double mutant is not significantly affected in long-distance systemic repression of these responses. Remarkably, we found that expression of many Pi-responsive genes is stimulated by sucrose in shoots and to a lesser extent in roots, and the sugar effect in shoots of Pi-starved plants was particularly enhanced in the cre1 ahk3 double mutant. Altogether, these results indicate the existence of multidirectional cross regulation between cytokinin, sugar, and Pi-starvation signaling, thus underlining the role of cytokinin signaling in nutrient sensing and the relative importance of Pi-starvation signaling in the control of plant metabolism and development.     

Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

Nitrogen Nutrition and Cytokinin Activity in Solanum tuberosum

In water culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of continuous nitrogen nutrition (constant supply of NO₃^?) and discontinuous nitrogen nutrition (interruption of NO₃^? supply, i. e., nitrogen withdrawal for 6 days) on the endogeneous cytokinin level in the roots, shoots and exudate of decapitated plants was studied. Harvests took place at intervals of 3 days. The chlorophyll formation test (cucumber cotyledons) and soya callus test were used to determine the cytokinin activity. With continuous nitrogen, the cytokinin activity decreased slightly with time in both roots and shoots but rose in the exudate. With discontinuous nitrogen, the nitrogen withdrawal led to a temporary, pronounced increase in cytokinin activity in the roots; at the same time, the cytokinin activity in the exudate decreased sharply. It is assumed that this temporary increase in cytokinin activity in the roots is a reflection of increased meristem activity in the roots. In the shoots, the cytokinin activity decreased during the nitrogen withdrawal period. These nitrogen-induced changes in cytokinin activity in the roots and shoots of potato should presumably have an important influence on the physiological age of the shoot, with all its consequences in the further development of the plant. Zeatin riboside was likely the main cytokinin component involved.     

Cytokinin: perception,signal transduction,and role in plant growth and development

Cytokinins are essential hormones for the proper growth and development of plants. They exert their actions through the phosphorylation of two-component signaling factors. The two-component elements in cytokinin signaling display not only overlapping, but also specific functions throughout a life cycle. These elements regulate the development of shoots, roots, and inflorescence meristems inArabidopsis; shoot meristems in rice; and nodule formation in the lotus. They are also involved in interactions between plants and pathogens. In this review, we examine the mechanism for signaling events initiated by cytokinins inArabidopsis.     

Cytokinin signaling

Cytokinins influence many aspects of plant growth and development. The current model for cytokinin signaling is a multi-step phosphorelay similar to the prokaryotic two-component systems that are used in responses to environmental stimuli. Recently, progress has been made in improving our understanding of the molecular mechanism that underlies cytokinin signaling. Molecular and genetic analyses of loss-of-function mutants indicate that the two-component elements that are involved in cytokinin signaling have redundant and overlapping functions. These elements regulate both the shoot and root meristems, are required for the development of fertile flowers, and modulate the response to varying nutrient levels.     

Hormone signaling in plant development

Hormone signaling plays diverse and critical roles during plant development. In particular, hormone interactions regulate meristem function and therefore control formation of all organs in the plant. Recent advances have dissected commonalities and differences in the interaction of auxin and cytokinin in the regulation of shoot and root apical meristem function. In addition, brassinosteroid hormones have recently been discovered to regulate root apical meristem size. Further insights have also been made into our understanding of the mechanism of crosstalk among auxin, cytokinin, and strigolactone in axillary meristems.     

Exogenous zeatin accumulation in wheat-root cells and its role in regulation of cytokinin transport

It was shown that the cytokinin content in the xylem sap of a wheat plant treated with exogenous zeatin was about ten times lower than in the nutrient solution in 24 h. Cytokinins were accumulated in roots rather shoots of treated plants. These data demonstrate the existence of a barrier in the cytokinin pathway from the nutrient solution to plant shoots. The deposition of lignin and suberin in stele detected with Sudan @III is enlarged with an increase in the distance from the tip of the root. The augmented content of suberin and lignin coincided with reduced cytokinin immunolabeling in root cells revealed by monoclonal antibodies to cytokinin and secondary gold-labeled antibodies. The accumulation of exogenous cytokinin in root stele cells shows that Casparian bands are not the only barrier on the cytokinin pathway to plant shoots. Intensive cytokinin immunolabeling in parenchyma cells surrounding stele vessels indicates the accumulation of cytokinin by these cells and suggests that there are mechanisms that limit the hormone loading in xylem vessels during transport to the shoot. The role of cytokinin transporters in this process is discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

植物不定芽离体再生分子调控的评述

植物的不定芽再生过程涉及众多基因及其互作。细胞分裂素诱导体细胞启动、启动的体细胞进行分裂和由此引发的茎分生组织发育是这一过程中的3个重要步骤。探讨这3个步骤的相关基因表达及其关系, 有助于揭示植物不定芽再生的分子调节机制。文章就这些步骤所涉及的分子调节过程的研究成果作一评述。     

Effect of Culture Origin and Conditions on Duvatrienediol Accumulation in Shoot Cultures of Tobacco

A comparison of morphology, trichome type and duvane accumulationof tobacco shoot cultures originating from seedlings, callusand cell suspension cultures incubated on a range of cytokininconcentrations is presented. A method for the extraction andHPLC analysis of duvatrienediols from cultures is described.Duvatrienediols (DVTs) were not detected in callus or cell suspensioncultures and were only detected in seedling-denved shoot cultures.Shoot development was dependent on explant type probably asa result of pre-exposure of callus and suspension cells to highauxin concentrations. Low cytokinin levels promoted shoot developmentwhilst high cytokinin levels increased the number of shootsregenerated but retarded their development. DVT accumulationoccurred only in ‘normally’ developed shoots withtrichomes. Growth of shoot cultures in liquid medium was inhibitoryto the accumulation of DVTs. Optimum conditions for the accumulationof DVTs were shown to be incubation of seedling-derived shootcultures on media containing 10^–6 M BAP solidified withagar. The yield was 120 µg g^–1 fr. wt. which iscomparable to greenhouse-grown material. Both and ß-DVTwere detected in a ratio of 5: 1. The relationship between tnchomesand DVT accumulation is discussed with reference to the rolesof culture origin, cytokinin, trichome type and environmentaleffects. Key words: Tobacco, duvatrienediol, trichome, shoot culture     

Organogenesis, Differentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenicand organogenic calli induced with nine different cytokinin/auxincombinations. Alkaloid colour reagents localized tropane alkaloidsin the vascular regions which had large cells in the secondaryxylem of the basal stem sections of the non-rooted shoots. Tropanealkaloids were localized in shoots regenerated from calli inducedwith two different cytokinin/auxin combinations. No alkaloidswere localized in shoots regenerated from calli induced withother cytokinin/auxin combinations. However, only nicotine wasdetected by GC-MS in the non-rooted shoots regenerated fromcalli induced with three different cytokinin/auxin combinations.Tropane alkaloids were also localized in xylem cells of rootsregenerated from calli induced with two different cytokininand auxin combinations independently. The presence or absenceof nicotine, hyoscyamine and scopolamine in different culturedplant materials was confirmed by GC-MS, indicating that althoughthe root is the main site for alkaloid biosynthesis, with suitablecell differentiation, alkaloid biosynthesis may take place incultured shoots without root initiation. Copyright 2000 Annalsof Botany Company Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate     

Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix

Although the effectiveness of topolins in plant tissue culture systems has recently been highlighted, there is a dearth of information on their interactions with auxins in relation to shoot organogenesis and secondary metabolite production. The current study evaluated the role of topolins singly or in combination with an auxin in comparison to 6-benzyladenine (BA) on shoot proliferation and secondary metabolite production of Huernia hystrix, a medicinal and ornamental stem-succulent of the endemic flora of southern Africa. Meta-topolin (mT) was more effective in improving shoot proliferation and phenolic production compared to BA. In general, the exogenous addition of α-naphthalene acetic acid (NAA) significantly increased shoot proliferation. The highest number of regenerated shoots (12.2 ± 0.98 shoots per explant) was recorded with medium containing 20 μM mT supplemented with 10 μM NAA and was three-times higher when compared to the treatments with cytokinin only. This suggests a synergistic interaction of auxin with cytokinin. On the other hand, supplementation with low NAA concentrations resulted in reduced in vitro flavonoid production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations (even when used in combination with NAA equimolar concentrations) significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on shoot proliferation and the production of secondary metabolites in H. hystrix.