N. V. Kudryakova

Cytokinin Stimulates Chloroplast Transcription in Detached Barley Leaves

Chloroplasts are among the main targets of cytokinin action in the plant cell. We report here on the activation of transcription by cytokinin as detected by run-on assays with chloroplasts isolated from apical parts of first leaves detached from 9-d-old barley (Hordeum vulgare) seedlings and incubated for 3 h on a 2.2 × 10−5 m solution of benzyladenine (BA). Northern-blot analysis also detected a BA-induced increase in the accumulation of chloroplast mRNAs. A prerequisite for BA activation of chloroplast transcription was preincubation of leaves for 24 h on water in the light, resulting in a decreased chloroplast transcription and a drastic accumulation of abscisic acid. Cytokinin enhanced the transcription of several chloroplast genes above the initial level measured before BA treatment, and in the case of rrn16 and petD even before preincubation. Cytokinin effects on basal (youngest), middle, and apical (oldest) segments of primary leaves detached from plants of different ages revealed an age dependence of chloroplast gene response to BA. BA-induced stimulation of transcription of rrn16, rrn23, rps4, rps16, rbcL, atpB, and ndhC required light during the period of preincubation and was further enhanced by light during the incubation on BA, whereas activation of transcription of trnEY, rps14, rpl16, matK, petD, and petLG depended on light during both periods. Our data reveal positive and differential effects of cytokinin on the transcription of chloroplast genes that were dependent on light and on the age (developmental stage) of cells and leaves.

Exogenous brassinosteroids activate cytokinin signalling pathway gene expression in transgenic Arabidopsis thaliana

Transgenic Arabidopsis thaliana plants carrying the GUS reporter gene fused to the promoter of the gene of primary response to cytokinins (CKs), ARR5, were used to estimate the influence of several brassinosteroids (BRs): brassinolide (BL), epibrassinolide (EBL), homobrassinolide (HBL), and 6-o-carboxymethyloxohomocastasterone (CHC) on the expression of CK signalling genes. BRs tested differed in their ability to activate the ARR5 gene promoter in 4-day-old seedlings and 3-week-old plants. BL caused the most prominent effect, yet it was considerably less than that of 6-benzylaminopurine (BA). An increase in GUS activity was observed in both dark and light conditions; however, the rate of elevation was higher in dark conditions. The activation of the PARR5:GUS fusion was accompanied by a moderate induction of the PAHK:GUS constructs, in which the reporter GUS gene was fused to the promoter of one of the CK receptor histidine kinases. The effects of BL on the AHK gene promoters were organ specific and correlated with the ability of a particular AHK gene to respond to BA treatment. BL activated the AHK3 promoter in 4-day-old seedlings and in shoots and roots of 3-week-old plants without any effect in detached leaves. The AHK2 gene promoter was activated by BA and BL only in seedlings, whereas the AHK4 gene promoter was activated only in roots. BL treatment caused the coordinate elevation of the CK levels in leaves to the same degree as the activation of the PAHK:GUS construct, suggesting that the accumulation of CKs was the reason for the activation by BRs of the CK signalling genes. The data obtained provide the evidence for the involvement of BRs in the regulation of the genes of the CK signalling pathway through an increase in the CK levels. However, the exact molecular mechanisms underlying BR-induced elevation of the CK content are unclear and warrant identification in the future.

Heat shock pre-treatment enhances the response of Arabidopsis thaliana leaves and Cucurbita pepo cotyledons to benzyladenine

The effects of heat shock (HS) pre-treatment on the response tobenzyladenine were studied in two plant model systems (1) retardation ofsenescence of Arabidopsis thaliana L. Heyhn rosette leavesand (2) induction of greening of detached Cucurbita pepoL.cotyledons. N6-benzyladenine (BA) retarded senescence of rosetteleaves of Arabidopsis thaliana (L) Heyhn and briefpre-treatment with HS (3 at 37 )essentially enhanced this cytokinin effect. BA stimulated cotyledon greening inCucurbita pepo L due to the activation of chlorophyllsynthesis. Brief cotyledon pre-heating at moderate temperatures (3 at 33–35 ) also enhanced thiscytokinin effect.

Membrane receptors of cytokinin and their regulatory role in Arabidopsis thaliana plant response to photooxidative stress under conditions of water deficit

Mild photooxidative stress combined with water deficit affected the expression of genes of cytokinin (CK) signal transduction. According to qRT-PCR data, these stress factors suppressed the expression of CK receptor genes AHK2 and AHK3 and the ARR5 response regulator gene, and slightly activated the expression of AHK4 gene. The absence of AHK2 and AHK3 receptors in Arabidopsis thaliana (L.) Heynh. mutants affected markedly the content of low-molecular-weight antioxidants (anthocyanins, carotenoids, proline) and resulted in the CK-dependent changes in the expression of genes, markers of oxidative stress, including RAB18, CHS, P5CS1, and PRODH1, under both normal and stressful conditions. At the same time, knockout of a single receptor gene or their combination did not induce CK-specific changes in the stress-activated expression of AOX1a, the marker gene of oxidative stress. CK receptor mutants lacked a statistically significant genotype influence on the photosystem II and photosystem I fluorescence yield under normal and stressful conditions. However, the system of CK perception was shown to be involved in the transcription regulation of the genes of the photosynthetic apparatus, ELIP2 and PSBS. Thus, CKs play an important role in plant responses to the photooxidative stress. The inactivation of the CK signaling system components proves to be one of the main strategies of Arabidopsis plant adaptation to the high light under conditions of water deficit.

Is a 67-kD Cytokinin-Binding Protein from Barley and Arabidopsis thaliana Leaves Involved in the Leaf Responses to Phenylurea Derivatives? (A Review)

Cytokinin-binding proteins (67-kD CBP) were isolated from barley first leaves of 8–10-day-old plants and Arabidopsis thaliana rosette leaves of 9-week-old plants. A capability of these proteins for cytokinin binding was assessed from trans-zeatin competition with antiidiotypic antibodies (ABa-i) for complex formation with immobilized 67-kD CBP under competitive ELISA conditions. ABa-i were obtained against anti-trans-zeatin antibody. 67-kD CBP from A. thaliana and barley leaves, in combination with trans-zeatin, activated transcriptional elongation in the in vitro system containing chromatin and RNA polymerase I from the same leaves. A phenylurea derivative demonstrating a high cytokinin activity, N-phenyl-N1-(2-chloro-4-pyridyl)urea (4-PU-30) could not displace ABa-i from its complex with 67-kD CBP from either barley or A. thaliana leaves, which indicates the absence of its interaction with CBP. In the presence of 67-kD CBP, 4-PU-30 did not activate transcription in the in vitro system containing chromatin and RNA polymerase I from A. thaliana or barley leaves. This means that, as distinct from trans-zeatin, 4-PU-30 did not use 67-kD CBP for transcription activation. A. thaliana leaf preincubation in the 4-PU-30 solution for 1 h enhanced RNA synthesis in the transcriptional system containing chromatin and RNA polymerase I from these pretreated leaves. Thus, leaves recognized the 4-PU-30 signal using another receptor. 4-PU-30 capacity to retard leaf senescence also supports this supposition. Another highly active phenylurea derivative thidiazuron also enhanced RNA synthesis in leaves and retarded their senescence.

Molecular and physiological responses of Arabidopsis thaliana plants deficient in the genes responsible for ABA and cytokinin reception and metabolism to heat shock

Using mutant plants of Arabidopsis thaliana, participation of the genes involved in abscisic acid (ABA) and cytokinins (CKs) metabolism and signaling in plant defense responses to heat shock (HS) was investigated. The magnitude of the stress action was assessed with biochemical indicators, such as accumulation of proline and malonic dialdehyde (MDA) and changes in the content of gene transcripts of heat shock proteins (HSPs) (HSP90.1 and HSP90.5) and transcription factor of HS (HSFA2) as well as stress-inducible genes, markers of oxidative stress (AOX1a, RD29, PRODH1, and P5CS1). Mutants with inactivated genes of ABA synthesis and, especially, signal perception exhibited lower thermo-resistance and accumulated elevated amounts of CK metabolism and signal transduction genes. In contrast, plants with inactivated components of CK synthesis and signal perception displayed increased tolerance to high temperatures and reduced levels of mRNA of oxidative stress genes as compared to wild type (WT) plants. However, enhancement of HS contributed to decrease of thermo-resistance of CKs receptor mutants up to the level of WT plants. Under hyperthermia, the stimulation of the plant defense mechanisms was accompanied by downregulation of the expression of CK metabolism and signal transduction genes (IPT3, CKX1, ARR5, AHK2, and AHK4) and ABA catabolism gene (CYP707A1) and upregulation of the expression of ABA synthesis and signal perception genes (ABA3 and ABI2). In the mutants insensitive to ABA, CYP707A1 gene was upregulated under the HS, while the expression of CK receptor gene expression did not reliably change. The results indicate that the response of the plants to elevated temperatures was determined not only by strength and the duration of the stress but the state of their ABA and CK metabolic and signaling systems as well.

Role of cytokinins in stress resistance of plants

The facts of both positive and negative influences of cytokinins on stress resistance of plants are known today. Without pretending to a final choice between these points of view, we have made an attempt to analyze the details of the experiments that gave rise to conclusions about the nature of the effect of cytokinins on the resistance to stress-causing influences with a focus on their intensity and duration. The review deals with the data concerning the influence of different adverse factors on the content of endogenous cytokinins and transduction of cytokinin signals, examines the influence on plant resistance of treatment with exogenous hormone, and the effects of genetic modifications causing changes in cytokinin content and signaling. Resistance is considered not only as a mean of plant survival under severe stress but also as an instrument of maintaining growth rate in plants exposed to moderate stress. Literature data and our own results make it possible to conclude that cytokinins play an important role in formation of plant resistance to adverse influences; however, the effect of these hormones depends on stress intensity. Under moderate stress, cytokinins ensure maintenance of plant growth, whereas a drop in cytokinins hampers growth under a strong influence of adverse factors, which is a prerequisite for mobilization of limited resources characteristic of severe stress and ensures preservation of plant viability.

Opposite roles of the Arabidopsis cytokinin receptors AHK2 and AHK3 in the expression of plastid genes and genes for the plastid transcriptional machinery during senescence

Key messageCytokinin membrane receptors of the Arabidopsis thaliana AHK2 and AHK3 play opposite roles in the expression of plastid genes and genes for the plastid transcriptional machinery during leaf senescenceAbstractLoss-of-function mutants of Arabidopsis thaliana were used to study the role of cytokinin receptors in the expression of chloroplast genes during leaf senescence. Accumulation of transcripts of several plastid-encoded genes is dependent on the АНК2/АНК3 receptor combination. АНК2 is particularly important at the final stage of plant development and, unlike АНК3, a positive regulator of leaf senescence. Cytokinin-dependent up-regulation of the nuclear encoded genes for chloroplast RNA polymerases RPOTp and RPOTmp suggests that the hormone controls plastid gene expression, at least in part, via the expression of nuclear genes for the plastid transcription machinery. This is further supported by cytokinin dependent regulation of genes for the nuclear encoded plastid σ-factors, SIG1-6, which code for components of the transcriptional apparatus in chloroplasts.

Effects of nitrate and ammonium on growth of Arabidopsis thaliana plants transformed with the ARR5::GUS construct and a role for cytokinins in suppression of disturbances induced by the presence of ammonium

Determination of physiologically active cytokinins in transformed Arabidopsis thaliana (L.) Heynh. plants (ecotype Wassilevskija) by means of the ARR5::GUS gene expression analysis and by the immunohistochemical assay demonstrated an increase of the cytokinin content during leaf growth and its decrease after growth cessation. After leaf growth cessation cytokinins (CKs) were found only in chloroplasts. Nitrate but not ammonium salt increased the cytokinin content in leaves. The replacement of nitrate in nutrient medium with ammonium salts suppressed leaf and root growth and decreased the content of physiologically active CKs in them. In plants cultivated on a media with NH4Cl as a sole nitrogen source, 10–9 M trans-zeatin several-fold activated leaf growth, prevented a decrease in the chlorophyll a and b content, and reduced proline accumulation indicating suppression of the NH4Cl stress action. Thus, negative effects of NH4Cl as a sole nitrogen source depended on the cytokinin shortage.

Effects of 24-epibrassinolide and green light on plastid gene transcription and cytokinin content of barley leaves ☆

HIGHLIGHTSEpibrassinolide and the green light up‐regulate the transcription of plastid genes.Epibrassinolide elevates the content of cytokinins in the dark.Green light increases the content of brassinosteroids and zeatin. ABSTRACT In order to evaluate whether brassinosteroids (BS) and green light regulate the transcription of plastid genes in a cross‐talk with cytokinins (CKs), transcription rates of 12 plastid genes (ndhF, rrn23, rpoB, psaA, psaB, rrn16, psbA, psbD, psbK, rbcL, atpB, and trnE/trnY) as well as the accumulation of transcripts of some photoreceptors (PHYA, CRY2, CRY1A, and CRY1B) and signaling (SERK and CAS) genes were followed in detached etiolated barley leaves exposed to darkness, green or white light ±1 &mgr;m 24‐epibrassinolide (EBL). EBL in the dark was shown to up‐regulate the transcription of 12 plastid genes, while green light activated 10 genes and the EBL combined with the green light affected the transcription of only two genes (psaB and rpoB). Green light inhibited the expression of photoreceptor genes, except for CRY1A. Under the green light, EBL practically did not affect the expression of CRY1A, CAS and SERK genes, but it reduced the influence of white light on the accumulation of CAS, CRY1A, CRY1B, and SERK gene transcripts. The total content of BS in the dark and under white light remained largely unchanged, while under green light the total content of BRs (brassinolide, castasterone, and 6‐deoxocastasterone) and HBRs (28‐homobrassinolide, 28‐homocastasterone, and 6‐deoxo‐28‐homocastasterone) increased. The EBL‐dependent up‐regulation of plastome transcription in the dark was accompanied by a significant decrease in CK deactivation by O‐glucosylation. However, no significant effect on the content of active CKs was detected. EBL combined with green light moderately increased the contents of trans‐zeatin and isopentenyladenine, but had a negative effect on cis‐zeatin. The most significant promotive effect of EBL on active CK bases was observed in white light. The data obtained suggest the involvement of CKs in the BS‐ and light‐dependent transcription regulation of plastid genes.