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The warming is global problem. In natural environments, heat stress is usually accompanied by drought. Under drought conditions, water content decreases in both soil and air; yet,the effect of lower air humidity remains obscure. We supplied maize and barley plants with an unlimited source of water for the root uptake and studied the effect of relative air humidity under heat stress. Young plants were subjected for 48 h to several degrees of heat stress: moderate (37 °C), genuine (42 °C), and nearly lethal (46 °C). The conditions of lower air humidity decreased the photochemical activities of photosystem I and photosystem II. The small effect was revealed in the control (24 °C). Elevating temperature to 37 °C and 42 °C increased the relative activities of both photosystems; the photosystem II was activated more. Probably, this is why the effect of air humidity disappeared at 37 °C; the small inhibiting effect was observed at 42 °C. At 46 °C, lower air humidity substantially magnified the inhibitory effect of heat. As a result, the maximal and relative activities of both photosystems decreased in maize and barley; the photosystem II was inhibited more. Under the conditions of 46 °C at lower air humidity, the plant growth was greatly reduced. Maize plants increased water uptake by roots and survived; barley plants were unable to increase water uptake and died. Therefore, air humidity is an important component of environmental heat stress influencing activities of photosystem I and photosystem II and thereby plant growth and viability under severe stress conditions.

The dark-detached leaves are widely used as a model to investigate senescence-delaying mechanisms of cytokinin (СК). The role of CK during senescence of detached Arabidopsis leaves in the light is less examined, although they provide unique opportunities for the study of the interaction of CK and the photosynthetic apparatus. Within the first four days of incubation of the leaves on water photosynthesis activity, chloroplast gene expression and chlorophyll content declined in all genotypes with a functioning CK receptor AHK3, while anthocyanin level and expression of its regulatory and biosynthesis genes increased. Application of exogenous CK visibly retarded the senescence symptoms but decreased photosystem II function, anthocyanin accumulation and promoted ROS production. The hormone stimulated starch accumulation in the chloroplasts and altered the expression of the gene for the extracellular cell wall invertase CWINV1. Although the AHK3 receptor played the most important role in the CK action, we also observed specific effects in loss-of function mutants for phosphortransmitters and type B response regulators. The results suggest that CK action in detached Arabidopsis leaves in the light is superimposed on modified carbon fluxes and starch turnover which results in an intricate combination of stress-related and senescence inhibiting effects.

Cd is one of the most toxic heavy metals and widespread pollutant. We searched for a direct Cd action on the photosynthetic electron transport chain using induced chlorophyll fluorescence and P700light absorption. Young barley and maize plants were treated with Cd in toxic (80 μM) and nearly lethal (250 μM) concentrations. The maximal and relative photochemical activities of PSI, its major limitation at the donor side, and partially acceptor-side limitation of PSII changed in agreement with Cd accumulation in the corresponding chloroplasts. Probably, acceptor-side limitation of PSII increased with a direct Cd action under 80 μM that was overcome with an indirect Cd action under 250 μM. These alterations can be explained by Cd/Cu substitution in plastocyanin. The photochemical and non-photochemical quenching by PSII varied diversely that cannot be explained unambiguously by any mechanism. The limitations of PSI (Y(ND), Y(NA)) and PSII (qC) were compared for the first time. They were ranged as follows: Y(NA) < qC < Y(ND). Short segments of qC and Y(ND) dynamics varied proportionally to each other. This implies the existence of an unknown mechanism adjusting limitations at the acceptor side of PSII (qC) and at the donor side of PSI (Y(ND)).HighlightsPSI activity changed in agreement with the changes of Cd content in chloroplastsThe data on PSII activity cannot be clearly explained by Cd actionPSII acceptor-side limitation qC was governed by opposed direct and indirect Cd actionsPSI and qC changes can be explained by Cd/Cu substitution in plastocyaninLimitations qC of PSII and Y(ND) of PSI changed proportionally for a short time

Senescence of leaves is initiated by differential expression of genes of transcription factors and is controlled by various phytohormones, among which cytokinins (CK), which negatively regulate this process, play an important role. According to the results of our transcriptome analysis and real-time PCR of selected genes, the delayed leaf senescence in mutants with an inactivated gene of CK receptor AHK2 was associated with altered expression of trans-factor genes belonging to the families AP2-EREBP, bHLH, C2H2, GATA, MYB, NAC, and WRKY. The most significant quantitative changes in the ahk2 mutants, as compared to the leaves of wild type plants, were observed in the expression profile of the bHLH38, bHLH39, and bHLH100 genes from the pool of iron uptake regulators. Significantly increased levels of transcripts of these genes were associated with the activation of the FEP2 (AT1G47395) and FEP3 (AT1G47400) genes, capable of inducing the expression of bHLH38 and bHLH39 under conditions of iron deficiency. The increased expression of the DREB26, ESE3, GATA4, ETC2, and F11O4.13 genes was accompanied by an increased accumulation of transcripts of the AGP17 gene of arabinogalactan protein as well as the IBL1, BEE2, Ole e 1, and GDSL genes associated with the regulation of cell wall proteins. Thus, the AHK2 receptor, usually is atributed with an auxiliary role in the perception of the CK signal by leaves, is associated with a specific set of genes involved in the regulation of iron outflow and degradation of cell wall components at the final stages of leaf ontogenesis.

Fine-tuned interactions between melatonin (MT) and hormones affected by environmental inputs are crucial for plant growth. Under high light (HL) conditions, melatonin reduced photodamage in

The expression profiles of the PAP genes, encoding proteins associated with plastid multisubunit RNA polymerase, were studied in dry seeds, during germination, and at the early stages of Arabidopsis thaliana seedling formation. A detailed analysis of the PAP transcript levels by RT-PCR showed that the transition of seeds from dormancy to active growth is accompanied by a drastic increase in the transcript accumulation of all studied genes on the first day of germination, both in the light and in the dark. Further changes in transcript levels differed among PAP genes and were apparently determined by their functional specificity. It was established for the first time that the expression of individual PAP genes is regulated by circadian rhythms, in addition to factors of ontogenetic and light nature.

A comparison was made of the physiological activity of brassinosteroids (BS) of different chemical structures—lactone-containing 24-epibrassinolide (EBL) and ketone-containing brassinosteroid 24-epicastasterone (ECS)—during dark aging of cut barley (Hordeum vulgare L.) leaves. The functional state of the photosynthetic apparatus, the amount of photosynthetic pigments, the content of lipid peroxidation products, and gene expression of HvS40 (senescence marker) were assessed. The separated barley leaves were incubated on solutions of EBL and ECS in the concentration range of 0.01–1 µM in Petri dishes on filter paper. Synthetic cytokinin 6-benzylaminopurine (BAP) at a 10 μM concentration was used as a positive control, and distilled water was used as a negative control. Petri dishes with leaves were kept in the dark for 5 days. Measurements of the analyzed parameters were carried out on the first, third, and fifth days from the beginning of incubation, mainly in the middle part of the leaf plate. ECS at a concentration of 1 μM reduced oxidative stress by the fifth day of incubation to a greater extent than EBL at a similar concentration. Regarding maintenance of chlorophyll a (Chl a) levels, 24-epicastasterone (1 μM) was more effective than 24-epibrassinolide. It was shown that exogenous brassinosteroids showed a protective effect on the main photochemical processes of the second photosystem during aging of barley leaves. Moreover, when assessing the coefficient of nonphotochemical quenching (qN), indicators of regulated (Y(NPQ)) and nonregulated (Y(NO)) energy dissipation, the ECS showed a more pronounced protective effect for the second photosystem compared to EBL. It was found that treatment with brassinosteroids reduced the level of gene expression of HvS40, a senescence marker on the fifth day of incubation, which was especially characteristic for the ketone-containing brassinosteroid. Thus, it can be concluded that the ketone-containing brassinosteroid not only exhibits high biological activity but, in terms of a number of parameters, is also more effective than the lactone-containing brassinosteroid in reducing the damage caused by aging.

Phytohormones are known to play an important role in the regulation of chloroplast biogenesis. However, information on the pathways of hormonal signal perception by chloroplasts is limited. In order to analyze the possible mechanisms of hormone action, the response to abscisic acid (ABA) and cytokinin (CKs) treatment of Arabidopsis thaliana (L.) Heynh. scabra3-2 (sca3-2) and rpotmp mutants with defective genes for nuclear-encoded plastid RNA polymerases was studied. The reaction of the rpotmp mutant usually did not differ from the response of wild-type plants: trans-zeatin activated the accumulation of transcripts of genes encoding the transcription apparatus and genes encoded by the plastid genome, while ABA weakly suppressed their expression or had no significant effect. However, the steady-state levels of gene templates of the plastid-encoded RNA polymerase (PEP-A) enzyme complex in the rpotmp mutant were higher than in wild-type plants, probably due to a compensatory mechanism that allows the mutant to enhance PEP-dependent transcription in the absence of the RPOTmp enzyme. Dysfunction of the plastid RNA polymerase RPOTp enzyme in the sca3-2 mutant induced an altered hormonal response of the genes for plastid transcriptional complex and plastid-encoded genes in comparison with the response of wild-type plants. The absence of a significant up-regulating effect of CKs on the expression of genes encoding the transcription apparatus correlated with the retention of the steady-state levels of transcripts for chloroplast genes and plastid proteins. In addition, the sca3-2 mutant had an increased steady-state expression of genes for CK metabolism: IPT3, IPT5, and CKX5. However, the response to the inhibitory effect of ABA in this mutant, on the contrary, was more pronounced, which is possibly caused by the changes in the steady-state levels of the transcripts of a number of genes encoding of ABA signaling and metabolism proteins. Thus, changes in the expression of NEP (nuclear-encoded RNA polymerases) genes under the action of hormones can be transformed into activation or inhibition in the expression of chloroplast genes of nuclear or plastome coding, providing the regulation of chloroplast functions through the mechanisms of anterograde signaling.

The theory of endosymbiotic origin of chloroplasts has become basal in present-day biology. In this regard, the emergence of eukaryotic photosynthesis has been established as a result of phagocytal capture of cyanobacteria by some ancestral eukaryotic cell. The phenomenon of endosymbiosis is an indispensable element of cell evolution theory. The process of endosymbiogenesis belongs to the crucial factors of biological progress that gave rise to astonishing diversity and complexity of the eukaryotic organisms. The modern theory of evolution is built on the joint base of the phenomena of endosymbiosis, parallel gene transfer, the theory of natural selection, and the data of phylogenomics. Ultimately, eukaryogenesis has integrated different supramolecular complexes in the cells, created the diversity of organelles, and has led to biosphere oxygenation and modern diversity of living forms.

Abstract De-etiolation or transition from etiolated growth (skotomorphogenesis) to photomorphogenesis is one of the most intriguing and intricate stages of plant ontogenesis. It comprises reprogramming of plant cell metabolism, reorganizing the operation of the hormonal system, and altering plant morphology. Dark growth in the soil mainly depends on phytohormones with gibberellins and brassinosteroids playing the leading role; on the soil surface, light as a major exogenous agent starts operating. It inhibits activity of the main repressor of photomorphogenesis (COP1) and regulators of transcription, which govern realization of gibberellin (DELLA) and brassinosteroid (BZR1/BES1) signals and activates trans-factors initiating transition to autotrophic nutrition (for instance, HY5). The strategy of etiolated growth consists in achieving a quick exposure to sunlight at the expense of active elongation of the stem. For transition to autotrophic nutrition, a plant must form a photosynthetic apparatus and protect itself from possible light injury. This review deals with the role of the main regulatory components ensuring etiolated growth and transition to photomorphogenic development.

Natural senescence of leaves is a regular physiological process subjected to a certain genetic program and controlled by various phytohormones. Cytokinins (CKs) play a crucial role in the leaf-senescence control being negative regulators of this process. We previously reported on the prolonged vegetative growth and the development of a double knockout ahk2ahk3 mutant of Arabidopsis thaliana Heynh. (L.) for the genes encoding CK receptors. According to the data of the transcriptomic analysis and real-time RT‑PCR of selected genes performed in this study, the delay of leaf senescence in ahk2ahk3 mutants is accompanied by enhanced (4–281-fold) expression of the group of SAUR (small auxin-up RNA) genes encoding auxin response proteins and by upregulation of the SSPP (senescence-suppressed protein phosphatase) gene, whose expression level significantly (137-fold) exceeded that in a wild-type transcriptome. Thus, it is quite possible that under significant inhibition of the CK signaling leaf senescence in Arabidopsis plants regulated via a compensatory mechanism connected with changes in the expression of the SAUR gene family and a certain pool of genes related to the auxin transport and signaling.

Chloroplasts' mechanisms of adaptation to elevated temperatures are largely determined by the gene expression of the plastid transcription apparatus. Gene disruption of iron-containing superoxide dismutase PAP4/FSD3 and PAP9/FSD2, which are parts of the DNA-RNA polymerase complex of plastids, contributed to a decrease in resistance to oxidative stress caused by the prolonged action of elevated temperatures (5 days, 30 °C). Under heat stress conditions, pap4/fsd3 and pap9/fsd2 mutants showed a decline in chlorophyll content and photosynthesis level, as measured by photosynthetic parameters, and a different amplitude of HSP gene response to heat stress. The expression of nuclear- and plastid-encoded photosynthesis genes and corresponding proteins was strongly inhibited in the mutants as compared with wild-type plants and was further suppressed or displayed no additional changes at 30 °C. NEP-dependent plastid genes, as well as NEP genes RPOTp and RPOTmp, were also downregulated in the mutants by high temperature or remained stable, unlike in wild-type seedlings where these genes were strongly upregulated. The results obtained correspond to the concept of the complex effect of various forms of reactive oxygen species under all types of stresses, including heat stress, and confirm the hypothesis of a new regulatory function in plastid transcription acquired by enzymatic proteins during evolution.

Eukaryotic photosynthesis originated in the course of evolution as a result of the uptake of some unstored cyanobacterium and its transformation to chloroplasts by an ancestral heterotrophic eukaryotic cell. The pigment apparatus of Archaeplastida and other algal phyla that emerged later turned out to be arranged in the same way. Pigment-protein complexes of photosystem I (PS I) and photosystem II (PS II) are characterized by uniform structures, while the light-harvesting antennae have undergone a series of changes. The phycobilisome (PBS) antenna present in cyanobacteria was replaced by Chl a/b- or Chl a/c-containing pigment–protein complexes in most groups of photosynthetics. In the form of PBS or phycobiliprotein aggregates, it was inherited by members of Cyanophyta, Cryptophyta, red algae, and photosynthetic amoebae. Supramolecular organization and architectural modifications of phycobiliprotein antennae in various algal phyla in line with the endosymbiotic theory of chloroplast origin are the subject of this review.

Fine-tuned interactions between melatonin (MT) and hormones affected by environmental inputs are crucial for plant growth. Under high light (HL) conditions, melatonin reduced photodamage in Arabidopsis thaliana and contributed to the restoration of the expression of the cytokinin (CK) synthesis genes IPT3, IPT5 and LOG7 and genes for CK signal transduction AHK2,3 and ARR 1, 4, 5 and 12 which were downregulated by stress. However, CK signaling mutants displayed no significant changes in the expression of CK genes following HL + MT treatment, implying that a fully functional cytokinin signaling pathway is a prerequisite for MT–CK interactions. In turn, cytokinin treatment increased the expression of the key melatonin synthesis gene ASMT under both moderate and HL in wild-type plants. This upregulation was further accentuated in the ipt3,5,7 mutant which is highly sensitive to CK. In this mutant, in addition to ASMT, the melatonin synthesis genes SNAT and COMT, as well as the putative signaling genes CAND2 and GPA1, displayed elevated transcript levels. The results of the study suggest that melatonin acts synergistically with CK to cope with HL stress through melatonin-associated activation or repression of the respective hormonal genes.

De-etiolation of plant seedlings is controlled by a complex system of light and hormonal signaling. This process is accompanied by chloroplast development and initiation of chlorophyll biosynthesis leading to cotyledon greening. In this study, peculiaritis of regulation of expression of the key genes encoding enzymes of chlorophyll biosynthesis in wild type plants and mutants of perception and transduction of blue light in Arabidopsis thaliana (L.) Heynh. during cytokinin (CK)-dependent greening of seedlings were investigated. Inactivation of blue-light signaling components (СRY1, CRY2 and HY5) led to retarded chlorophyll accumulation and decrease of the expression of the key genes responsible for its biosynthesis. Exogenous application of cytokinin accelerated de-etiolation of wild type and knockout-mutants cry1cry2 and hy5 seedlings that was manifested itself in elevating chlorophyll a fluorescence and higher level of transcripts of a number of genes involved in chlorophyll biosynthesis. Taken together the results may provide evidence for the significant contribution of blue-light signaling to the greening regulation at early steps of de-etiolation of seedlings and involvement of HY5 protein in realization of the positive effect of CK at early steps of photomorphogenesis.

Melatonin, a potent regulator during plant development and stress responses, affects diverse plastid-related processes. However, its role in the regulation of plastid gene expression is largely unknown. In this study, exogenous melatonin was shown to reduce the negative influence of excess light by increasing the efficiency of the photosystems and rearranging the expression of chloroplast- and nuclear-encoded genes in detached Arabidopsis leaves. The positive effects of melatonin predominantly occurred at lower concentrations, while high doses had an inhibitory effect. The impact of melatonin was not straightforward. It mainly influenced the expression of the genes encoding the chloroplast transcription machinery and housekeeping genes involved in maintaining transcriptional activity and the functional state of chloroplasts. Despite the fact that melatonin treatment improved photosynthetic parameters, the levels of photosynthesis gene transcripts and photosynthetic proteins remained practically unaltered suggesting that melatonin impact on photosynthetic apparatus which would allow the balancing of chloroplast functions with stress responses is highly complicated.

In silico analysis of the promoter region of the At4g01870 gene of Arabidopsis thaliana (L.) Heynh. showed the presence of ABRE, W-box, RAV1-A, MYB, and LFY cis-elements in the sequence. These regulatory motifs bind the transcription factors involved in responses to abscisic acid (ABA) and stresses. Stable transgenic plants carrying the β-glucuronidase gene under the control of the 5'-deletion fragments of the At4g01870 promoter were obtained. According to the results of histochemical staining of transformants, gene expression was induced by abiotic stress and was most significant in the conductive tissues of the root, leaves, and sepals as well as in flowers. The study of At4g01870 gene expression by RT-PCR confirmed that the gene transcript content increased after the exposure of plants to a solution of NaCl or at 37°C and after ABA treatment; however, hypothermia almost unchanged the level of accumulation of the transcripts. Along with ABA, expression of the At4g01870 gene was induced by indolylacetic and salicylic acids and ethylene precursor 1-aminocyclopropane-1-carboxylic acid; it was hardly regulated by methyl jasmonate and inhibited by cytokinin. The TolB-like protein, encoded by the At4g01870 gene, functions as a type of platform, based on which protein complexes are assembled. Given the previously identified ABA-binding properties of the protein At4g01870 and the presence of the ABA-dependent cis-elements in the promoter of its coding gene, it can be assumed that the protein encoded by the At4g01870 gene allows to control the hormonal signals in the cell, providing a structural platform for the interaction of specific effector proteins, trans-factors and ion channels.

Cytokinins (CKs) are known to regulate the biogenesis of chloroplasts under changing environmental conditions and at different stages of plant ontogenesis. However, the underlying mechanisms are still poorly understood. Apparently, the mechanisms can be duplicated in several ways, including the influence of nuclear genes that determine the expression of plastome through the two-component CK regulatory circuit. In this study, we evaluated the role of cytokinins and CK signaling pathway on the expression of nuclear genes for plastid RNA polymerase-associated proteins (PAPs). Cytokinin induced the expression of all twelve Arabidopsis thalianaPAP genes irrespective of their functions via canonical CK signaling pathway but this regulation might be indirect taking into consideration their different functions and versatile structure of promoter regions. The disruption of PAP genes contributed to the abolishment of positive CK effect on the accumulation of the chloroplast gene transcripts and transcripts of the nuclear genes for plastid transcription machinery as can be judged from the analysis of pap1 and pap6 mutants. However, the CK regulatory circuit in the mutants remained practically unperturbed. Knock-out of PAP genes resulted in cytokinin overproduction as a consequence of the strong up-regulation of the genes for CK synthesis.

The physiological and molecular responses of Arabidopsis thaliana plants to cold stress were studied. Exposure to a low non-freezing temperature (4°C, 5 days) caused a decrease in the physiological functions and activity of a number of photosynthetic genes and elevation in expression of the cold stress gene COR15a, the product of which protects chloroplasts. It was shown for the first time that in parallel to a general inhibition of physiological functions under hypothermia, an increase in the expression of most genes for the chloroplast transcription apparatus was observed. This is obviously one of the compensatory mechanisms of adaptation aimed to maintain cellular homeostasis and physiological functions under hypothermia.

HY5 (ELONGATED HYPOCOTYL5), a bZIP transcription factor, is one of the main regulators of light and hormonal signaling. Among the targets of this gene, the genes for the transcriptional complex of chloroplasts whose coordinated expression ensures the initial stages of photomorphogenesis are particularly significant. In this study, we showed that, during de-etiolation, HY5 functions as a positive CK-dependent regulator of the expression of genes encoding proteins associated with plastid RNA polymerase (PAP), which functions below the primary chain of sensing the cytokinin signal. The absence of blocking effect of mutations of the CRY1, CRY2, PHYA, and PHYB photoreceptor genes on the CK-dependent content of PAP gene transcripts indicates the parallel action of the hormone and light in their regulation.

Molecular mechanisms of cytokinin effect as the key activators of chloroplast biogenesis have been thoroughly investigated in recent decades; however, the role of this class of phytohormones in the regulation of expression of the plastome transcription machinery genes is obscure. In order to look into the effect of the components of the cytokinin signal system on plastid transcription machinery during deetiolation, we analyzed light- and cytokinin-dependent expression dynamics of chloroplastic RNA-polymerases, PAP proteins, and transcription factors genes upon light exposure of 4-day-old seedlings of wild type Arabidopsis thaliana (L.) Heynh. (Columbia-0) and knockout mutants for perception and transduction of the cytokinin signal. Both agents exerted a selective influence on the expression of different genes of the plastome transcription machinery. The positive effect of light and cytokinin on deetiolation probably depended on the operation of receptors AHK3 and AHK4 and response regulator genes АRR1, АRR10, and ARR12.

During the last two decades much success in studying plastid genome structure and expression was achieved. The primary sequence of hundreds of plant plastid genomes has been determined, which allows us to understand basic laws of plastome structure. Novel chloroplast nuclear-encoded RNA-polymerases and sigma-factors have been discovered. Mechanisms responsible for posttranscriptional regulation of plastid genes' expression, including splicing and editing, are actively being studied. Increasingly more data on the most important role of nucleoids in chloroplast biogenesis is emerging. Much attention is presently given to studying the proteins associated with bacterial type RNA-polymerase. Determination of the primary sequence of genomes for a number of higher plants has produced new information on the exchange of genetic material between cell organelles. At present, interorganelle signaling in plant cells is a subject of active study. In the review, a wide range of issues is quite briefly discussed, including some elements of chloroplast evolution and nuclear-plastid signaling. Certain features of chloroplast genetic material organization are described. In addition, basic stages of plastid genome expression are discussed.

Cytokinins (CKs) have a strong influence on plastid development. We used the Arabidopsis thaliana (L.) Heynh. loss-of function insertion mutants ahk2, ahk3 and ahk4 alone or in different combinations to examine the effect of individual membrane cytokinin (CK) receptors on the expression of plastid genes and nuclear genes for the plastid transcription machinery. Quantitative real-time PCR-analyses demonstrate that the CK membrane receptors have overlapping functions in regulating the levels of plastid house-keeping genes in 7-day-old seedlings, relative to the nuclear UBQ10 gene. In contrast, photosynthesis genes responded differently to AHK3 and AHK2 receptor inactivation and the results show a dominant role of the AHK2/AHK3 receptor combination in the CK responses. These differences could be, at least in part, mediated through the regulation by the nuclear-encoded RPOTp and RPOTmp genes for plastid phage-type RNA polymerases. CK positively regulated the transcript abundance of the nuclear encoded SIG2 and SIG6, and negatively that of SIG4 and SIG5 for sigma factors of the plastid transcription apparatus. We propose that these two sigma factors may affect the CK-controlled plastid gene expression.

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.

Cd, Cu, and Fe were used to reveal the specificity of their toxic actions. We studied the effects of heavy metals on the growth of barley seedlings, contents of cations in leaves and chloroplasts, induced chlorophyll fluorescence and P700 light absorption. Differences were found at each level of research. We measured the contents of Cd, Cu, Fe, Mn, Zn, Ca, Mg, and K. The proportion of cations in leaves targeted to chloroplasts varied from 0.1% (K) to >90% (Fe). Their levels changed in different ways. We found no correlation between changes in cation contents in leaves and chloroplasts. Treatment with Cd, Cu, and Fe increased the contents of some cations. The extra portions were targeted primarily out of chloroplasts, which was most noticeable in the case of Cu and Fe. Cd treatment decreased non-photochemical quenching with concomitant increases in closed photosystem II. We introduced new coefficients qC for closed photosystem II and X(II) to compare the yields of photosystem II and photosystem I. Cd likely decreased both PSI content in leaves and its quantum yield. In control plants, the quantum yield ratio of PSI/PSII increased gradually from 1.25 under low light to 4 under high light. Cd treatment prevented the increase under moderate light; under high light the ratio reached 2. Cu treatment increased the acceptor side limitation of photosystem I under low light; components of the Calvin cycle likely demand more light for activation in Cu-treated plants.

Analysis by real-time PCR of single and double insertion mutants of A. thaliana with inactivated cytokinin receptor genes showed that the level of transcripts of some of plastid genes during skotomorphogenesis depended on the state of functionally active receptor AHK3. The cytokinin-regulated plastid encoded genes involved the housekeeping genes (rpoB and accD) and the genes for photosynthetic proteins (ndhA, psbA, atpB, and psaA). However, the absence of hormone activation of plastid encoded genes engaged in the translation of plastid proteins, rRNA (rrn16), and tRNA (trnE), indirectly indicate the disruption of the synthesis of chloroplast proteins in the dark.

The treatment of Arabidopsis thaliana plants with exogenous cytokinin (CK) followed by heat shock (HS) activated the expression of the genes for the plastid transcription machinery but adversely affected the plant viability. Abscisic acid (ABA), conversely, promoted maintaining the resistance to HS and had differentially affected different components of the plastid transcriptional complex. This hormone suppressed the accumulation of transcripts of PEP genes and the genes encoding PAP proteins, which are involved in DNA-RNA metabolism. However, it had no effect or activated the expression of NEP genes and PAP genes, which are involved in the redox regulation, as well as the genes encoding the stress-inducible trans-factor (SIG5) and the plastid transcription Ser/Thr protein kinase (cpCK2). Thus, for the adaptation of plants to elevated temperatures, both increase and decrease in the expression of the genes for the plastid transcriptional machinery with the involvement of various regulatory systems, including phytohormones, are equally significant.

Plant growth and photosynthetic activity are usually inhibited due to the overall action of Cd on a whole organism, though few cadmium cations can invade chloroplasts in vivo. We found that in vivo, the major portion of Cd in barley chloroplasts is located in the thylakoids (80%), and the minor portion is in the stroma (20%). Therefore, the electron-transport chain in the thylakoids would be the likely target for direct Cd action in vivo. In vitro, we found the distribution of Cd to be shifted to the stroma (40–60%). In barley chloroplasts, the major portions of Mg, Fe, Mn, and Cu were found to be located in the thylakoids, and most Ca, Zn, and K in the stroma. This finding was true for both control and Cu- or Fe-treated plants. Treatment with Cd affected the contents of all cations, and the largest portions of Ca and Zn were in the thylakoids. Alterations of the K and Mn contents were caused by Cd, Cu, or Fe treatment; the levels of other cations in chloroplasts were changed specifically by Cd treatment. The quantity of Cd in chloroplasts was small in comparison to that of Mg, Ca, and Fe. In thylakoids, the amount of Cd was similar to that of Cu and comparable to the levels of Zn and Mn. Accordingly, the possible targets for direct Cd action in thylakoids are the Mn cluster, plastocyanin, carbonic anhydrase, or FtsH protease. The quantity of Cd in thylakoids is sufficient to replace a cation nearly completely at one of these sites or partially (20–30%) at many of these sites.

Heat shock is one of the major abiotic factors that causes severe retardation in plant growth and development. To dissect the principal effects of hyperthermia on chloroplast gene expression, we studied the temporal dynamics of transcript accumulation for chloroplast-encoded genes in Arabidopsis thaliana and genes for the chloroplast transcription machinery against a background of changes in physiological parameters. A marked reduction in the transcript amounts of the majority of the genes at the early phases of heat shock (HS) was followed by a return to the baseline levels of rbcL and the housekeeping genes clpP, accD, rps14 and rrn16. The decline in the mRNA levels of trnE (for tRNAglu) and the PSI genes psaA and psaB was opposed by the transient increase in the transcript accumulation of ndhF and the PSII genes psbA, psbD, and psbN and their subsequent reduction with the development of stress. However, the up-regulation of PSII genes in response to elevated temperature was absent in the heat stress-sensitive mutants abi1 and abi2 with the impaired degradation of D2 protein. The expression of rpoA and rpoB, which encode subunits of PEP, was strongly down-regulated throughout the duration of the heat treatment. In addition, heat stress-induced PEP deficiency caused the compensatory up-regulation of the genes for the nuclear-encoded RNA polymerases RPOTp and RPOTmp, the PEP-associated proteins PAP6 and PAP8, the Ser/Thr protein kinase cPCK2, and the stress-inducible sigma factor gene SIG5. Thus, heat stress differentially modulates the transcript accumulation of plastid-encoded genes in A. thaliana at least in part via the expression of HS-responsive nuclear genes for the plastid transcription machinery.

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.

We characterized AA1 (Abscisic acid Activated 1), a protein from Lupinus luteus L. predicted to be located in the apoplastic space who’s mRNA and protein levels are strongly regulated by ABA, salt stress, and hypothermia. A fragment from the recombinant AA1 protein binds ABA as shown by the spectrofluorimetric titration assay of the protein by ABA. The BLAST software of the DFCI database identified more than 200 ESTs from 46 dicots and monocots, including three genes with unknown function from Arabidopsis thaliana, which are closely related to the lupine AA1. The central part of the proteins encoded by these genes contains the TolB motif from Escherichia coli and shares conserved WD40-like repeats, which form the basis for the tertiary beta-propeller structure and provide a potential platform for the assembly of protein complexes. Our data suggest that the highly conserved AA1 proteins from L. luteus and other higher plants are involved in ABA-mediated responses.

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.

It was shown for the first time the presence of the soluble cytokinin-binding protein with a mol wt of 67 kD (CBP67) in the cyanobacterium Synechocysti sp. PCC 6803. CBP67 was isolated by two independent methods: by affinity chromatography on zeatin-Sepharose and by immunoaffinity chromatography on zeatin-Sepharose using monoclonal antibodies (mAb) raised against maize CBP70 (70 kD). The cytokinin-binding properties of CBP67 were assessed by its interaction with anti-idiotype Ab (Aba-i) raised against Ab to zeatin. Aba-i are, in fact, Ab to zeatin-binding site of the protein. The high specificity of interaction between trans-zeatin and CBP67 was demonstrated. Zeatin riboside and cis-zeatin mahifested a reduced interaction with CBP67; other phytohormones (auxin, gibberellic acid, and ABA) and adenine did not interact with the protein at all. Cyanobacterial CBP67 activated in vitro transcription in the Synechocystis lysate. The highest effect on transcription was observed at a combined action of CBP67 and trans-zeatin. Cyanobacterial CBP67 activated transcription in the barley chloroplast lysate as well. The totality of the results indicates a possibility of the existence of systems of cytokinin signal perception in the evolutionary precursor of chloroplasts, cyanobacteria, which could bring this system into a plant cell.

The effects of salicylic acid (SA) on the rate of respiration and the activity of cyanide-resistant sensitive to salicylhydroxamic acid oxidation pathway in detached etiolated cotyledons of yellow lupine (Lupinus luteus L.) and mitochondria isolated from these cotyledons were studied. Cotyledon treatment with 1 mM SA for 12 h increased the rate of oxygen uptake predominantly due to the activation of cyanide-resistant respiration (CRR) and alternative pathway of mitochondrial oxidation. It was established that the lupine genome encodes at least two isoforms of alternative oxidase (AO), LuAOX1 and LuAOX2, with the mol wt of about 35 kD. These proteins are always present in the mitochondria of etiolated lupine cotyledons, but their level increased rapidly after cotyledon treatment with SA, probably by increasing the mRNA content of the corresponding genes. SA-induced expression of Aox genes was correlated with the activation of CRR and an increase in the maximal activity (capacity) of AO in both detached yellow lupine cotyledons and mitochondria isolated from them.

Numerous studies have shown effects of abscisic acid (ABA) on nuclear genes encoding chloroplast-localized proteins. ABA effects on the transcription of chloroplast genes, however, have not been investigated yet thoroughly. This work, therefore, studied the effects of ABA (75 μM) on transcription and steady-state levels of transcripts in chloroplasts of basal and apical segments of primary leaves of barley (Hordeum vulgare L.). Basal segments consist of young cells with developing chloroplasts, while apical segments contain the oldest cells with mature chloroplasts. Exogenous ABA reduced the chlorophyll content and caused changes of the endogenous concentrations not only of ABA but also of cytokinins to different extents in the basal and apical segments. It repressed transcription by the chloroplast phage-type and bacteria-type RNA polymerases and lowered transcript levels of most investigated chloroplast genes drastically. ABA did not repress the transcription of psbD and a few other genes and even increased psbD mRNA levels under certain conditions. The ABA effects on chloroplast transcription were more pronounced in basal vs. apical leaf segments and enhanced by light. Simultaneous application of cytokinin (22 μM 6-benzyladenine) minimized the ABA effects on chloroplast gene expression. These data demonstrate that ABA affects the expression of chloroplast genes differentially and points to a role of ABA in the regulation and coordination of the activities of nuclear and chloroplast genes coding for proteins with functions in photosynthesis.

Cytokinin 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 senescence Loss-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.

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 ±1μm 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.

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.

The expression regulation by cytokinin of genetic constructs PAHK2-GUS, PAHK3-GUS, and PAHK4-GUS in transgenic Arabidopsis thaliana (L.) Heynh plants bearing the gene encoding β-glucuronidase (GUS) under the control of the promoter of one of three genes encoding histidine protein kinases, which are membrane receptors of cytokinin was studied. In 4–5-day-old etiolated A. thaliana seedlings, treatment with cytokinin resulted in the strongest expression activation of the constructs PAHK2-GUS and PAHK3-GUS. The same constructs were activated by cytokinin also at the seedling transit from scoto- to photomorphogenesis. Long-term seedling growing in darkness on medium containing cytokinin resulted in the substantial promoter activation of the gene encoding the histidine kinase AHK2. In the leaves of three-week-old plants with actively functioning chloroplasts, treatment with cytokinin mainly stimulated expression of the construct PAHK3-GUS. In detached senescing leaves, treatment with cytokinin retarded the loss of chlorophyll but did not affect significantly GUS activity under both light and darkness conditions in either of tested lines containing GUS gene under the control of promoters of histidine kinase genes. At the same time, cytokinin activated the promoter of the gene of primary response to cytokinin in the construct PARR5-GUS. Thus, in the studied test-system, treatment with cytokinin of A. thaliana plant grown in darkness or in the light affected differently the expression of histidine kinase genes in dependence of plant age, conditions of plant cultivation, and plant physiological state.

Comparative analysis of growth characteristics, content of auxins and cytokinins, and the level of photosynthetic pigments was conducted in det2 mutant plants of Arabidopsis thaliana (L.) Heynh notable for a disturbed synthesis of brassinosteroids and therefore their low endogenous level. In this mutant line, we observed de-etiolation: the formation of large cotyledons and long hypocotyls in the dark already shown earlier. For the first time, we analyzed transcription of 12 chloroplast genes encoding functionally different proteins and RNAs in rosette leaves of wild type and det2 plants. In rosette leaves of mutant plants, transcription activity of the investigated plastid genes was much higher than in the parental line. On these grounds, we assumed that the low level of brassinosteroids was correlated with the activation of transcription of some chloroplastic genes and realization of the de-etiolation program.

Plastids are evolutionary descendants of cyanobacteria and retain many proteins of cyanobacterial origin including histidine kinases (HKs). Histidine kinases form a major group of protein kinases in cyanobacteria but a minor group in angiosperms; no HK has been detected in plant plastids so far. This raises the question: have higher plant plastids retained some cyanobacterial His/Asp regulatory systems or the latter have been replaced with Ser/Thr or Tyr protein kinases more typical for eukaryotes? To answer this question H1 antiserum against conservative phospho-acceptor motif of HKs was raised and applied for the analysis of different chloroplast fractions. In maize, three polypeptides with apparent molecular masses about 26, 27.5, and 28 kDa were revealed in thylakoid membranes. The results of in organello phosphorylation suggest that these proteins may be His-phosphorylated. Polypeptides with similar molecular masses were revealed in various mono- and dicotyledonous plants. Bioinformatic analysis demonstrated that in angiosperms these polypeptides might result from alternative transcription initiation and/or alternative processing of mRNAs of genes encoding well-known HKs. Besides, the genome of the moss Physcomitrella patens contains much more genes that could code for plastid HKs. The data obtained let us suppose that plastids of contemporary plants have HK(s) that could be involved in regulation of plastid gene expression.

The effects of Cd, Cu, and Ni ions on chloroplast gene expression were studied in 7-day-old barley (Hordeum vulgare L.) seedlings. Toxicity of heavy metals (HM) was estimated from their effects on root and shoot growth and from the accumulation of HM in roots and leaves of seedlings grown in roll culture. Root growth was affected by HM to a higher extent than shoot growth. A statistically significant inhibition of root growth was observed at concentrations of all studied HM ≥100 μM. Leaf growth was inhibited by Cu and Ni at concentrations ≥400 μM, while the inhibitory effect of Cd on leaf growth was observed at concentrations ≥100 μM. The translocation of Cu from roots to shoots of barley seedlings proceeded slower than the accumulation of Cu in roots. Unlike Cu, Ni was actively accumulated by shoots in addition to its strong absorption by roots. Using the run-on transcription and RT-PCR (reverse transcription-polymerase chain reaction), we showed that HM at a concentration of 100 μM regulated the expression of individual chloroplast genes. The presence of either Cd, Cu, or Ni ions in the plant growth solution enhanced transcription of rpl16, rpl23, and rpl12 genes encoding ribosomal proteins, and of ndhA encoding the subunit 1 of plastid NADH-plastoquinone oxidoreductase. Transcription of sixteen other studied plastid genes was not affected by HM. The activation of transcription of the studied genes was not always accompanied by an increase in the transcript copy number. Northern blot analysis showed that Cd impaired the splicing of rpl16, a chloroplast gene that contains an intron. Thus, this study proves that HM regulate not only the transcriptional activity of chloroplast genes but also the splicing of rpl16 transcripts.

The main stages of the run-on transcription method as applied to the quantification of mitochondrial gene transcription are described. As distinct from widely applied methods of northern hybridization and PCR after reverse transcription, this method permits studying the effects of exogenous and endogenous factors on the rate of mitochondrial gene transcription. The main method stages are (1) isolation of intact organelles; (2) preparation of nylon membranes with gene-specific fragments of DNA; (3) transcription in the lysates of these organelles in the presence of 32P-labeled RNA precursors; and (4) hybridization of newly synthesized labeled transcripts with DNA fragments of tested mitochondrial genes loaded on the nylon membrane.

The regulatory role of cytokinins and abscisic acid (ABA) during de-etiolation of monocotyledonous plants is not sufficiently studied. The effects of cytokinin—6-benzyladenine (BA) and ABA on leaf greening and chloroplast gene transcription were studied on leaves detached from etiolated barley (Hordeum vulgare L.) seedlings. The influence of both phytohormones was age-dependent. Cytokinin practically had no effect on the youngest (3-day-old) leaves, increased chlorophyll accumulation in 6-day-old leaves, and this effect decreased in 9-day-old leaves. ABA reduced chlorophyll accumulation in the youngest leaves, and this effect was blocked by cytokinin. ABA alone had practically no effect on greening of leaves detached from 6- and 9-day-old seedlings, but fully inhibited activation of this process by cytokinin. Thus, antagonism between ABA and cytokinin was observed for seedlings of all ages. The rate of transcription of chloroplast genes was studied by run-on transcription in isolated chloroplasts in the course of etiolated seedling growth in darkness, and age-dependent decrease was demonstrated. It was established that cytokinin activated transcription of 11 chloroplast genes among 30 studied, and ABA suppressed transcription of 12 plastid genes. Such differential effect of cytokinin and ABA on chloroplast gene transcription in monocot seedlings during etiolated leaf greening was not known before.

The effects of phytohormones on the activity of lipoxygenase (LOX) and the content of 9-LOX mRNAs in potato (Solanum tuberosum L., cv. Odissei) mini-tubers were studied in model (incubation of disks cut from mini-tubers on phytohormone solutions) and vegetation (spraying with phytohormone solutions at various developmental stages) experiments. In model experiment, it was shown that most tested phytohormones (auxin, kinetin, epibrassinolide, gibberellin, and salicylic acid) suppressed LOX activity. At the same time, auxin and gibberellin increased the amount of 9-LOX gene transcripts, whereas other phytohormones reduced it. Under conditions of vegetation experiment, principal differences between auxin and kinetin action were revealed. In both types of experiments, kinetin affected similarly both LOX activity and the amount of 9-LOX transcripts. In the case of auxin treatments, opposite situation was observed: the highest content of 9-LOX gene transcripts corresponded to the lowest enzyme activity. The results obtained show a great phytohormone influence on different stages of 9-LOX gene expression. Kinetin affected enzyme activity via its effect on the level of transcription, whereas auxin controls LOX activity on another level, although it affects the content of transcripts as well.

We suggest a design of experiment, which permits studying a differential regulation of chloroplast gene transcription by exogenous cytokinin, using detached first barley leaves. Such experiments are of great interest for investigation of molecular mechanisms of the chloroplast biogenesis hormonal regulation. It was demonstrated that a cytokinin control of plastid genome transcription depended on the plant age, a source of detached leaves, chloroplast position in the leaf blade, and the intensity and duration of leaf illumination. Transcription regulation was studied for the first barley leaves detached from 9-day-old barley plants grown in soil. The leaves were preincubated for 24 h in the light (270 μmol/(m2 s) and then transferred for 3 h on water or cytokinin solution (benzyladenine, 2.2 × 10−5 M) under similar lighting condition. Chloroplasts were isolated from the most sensitive to cytokinin apical leaf part and used for run-on transcription. From 26 tested genes, cytokinin activated the rate of transcription of 19 genes (by 2 to 9 times as compared with control) and did not affect transcription of 7 genes. Cytokinin-sensitive genes encode proteins of photosynthesis and the components of the plastid genome expression system. The results obtained allow transition to more detail studying the mechanisms of hormonal regulation of chloroplast genome transcription.

A new approach for maize genetic transformation using Agrobacterium tumefaciens was developed. It allows the usage of both immature embryos and embryogenic calluses as explants. This method includes long-term (15–20 days) cultivation of maize tissues on the agrobacterial monolayer. In this case, necrotic lesions did not appear and all cells were alive. Regenerated plants expressed genes for neomycine phosphotransferase and β-glucuronidase. The presence of the nptII gene was confirmed by PCR analysis and Southern-blot hybridization. In dependence on maize genotype and experimental conditions, the frequency of genetic transformation varied from 1.5 to 16%. Inheritance of genes introduced was demonstrated. The method elaborated could be applied to a wide range of maize genotypes.

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10−6 M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.