rus
DOI: 10.22184/1993-7296.FRos.2020.14.2.192.210
The article deals with the issues of light regulation of the plant genetic system and light control of morphogenesis. The idea of the mechanisms of light signal translation in the cell is given. The relationship between photoreceptor proteins and endogenous programs of plant development
is shown. The role of pigment proteins and phytohormones in the regulation of plant
ontogenesis is characterized. Experimental results demonstrating light control of plant
morphogenesis are presented.
The article deals with the issues of light regulation of the plant genetic system and light control of morphogenesis. The idea of the mechanisms of light signal translation in the cell is given. The relationship between photoreceptor proteins and endogenous programs of plant development
is shown. The role of pigment proteins and phytohormones in the regulation of plant
ontogenesis is characterized. Experimental results demonstrating light control of plant
morphogenesis are presented.
REGULATING EFFECT OF LIGHT ON PLANTS
Yu. N. Kulchin1,2, D. O. Goltsova1, E. P. Subbotin1
Institute of automation and control processes FEB RAS
Far Eastern Federal University, Vladivostok, Russia
The article deals with the issues of light regulation of the plant genetic system and light control of morphogenesis. The idea of the mechanisms of light signal translation in the cell is given. The relationship between photoreceptor proteins and endogenous programs of plant development is shown. The role of pigment proteins and phytohormones in the regulation of plant ontogenesis is characterized. Experimental results demonstrating light control of plant morphogenesis are presented.
Keywords: light, light control, spectrum, pigment proteins, photoreception, phytohormones, plants ontogenesis.
Received: 09.09.2019
Accepted: 07.10.2019
INTRODUCTION
Sunlight is an important adaptive stimulus, and many living organisms adapt their metabolism to the conditions of variable lighting in the environment, perceiving and reacting to light signals by changing their physiological functions [1]. In this case, light acts as a multifaceted factor characterized by qualitative (range of wavelengths) and quantitative (intensity, integral daily radiation, photoperiod) parameters, as well as direction and polarization. As a rule, the main reason for the low productivity of plants growing under natural conditions is that a huge amount of energy coming from the Sun is depreciated as a factor in photosynthesis due to unfavorable combinations of lighting parameters with other productivity conditions: heat, humidity, and soil fertility conditions [2]. Given that light controls the functioning of endogenous regulation systems (gene, enzymatic, trophic, hormonal, etc.), the combined effect of which provides an adequate response of plants to lighting conditions, by manipulating the characteristics of lighting, it is possible to maximize the potential determined by the genetic plan of the plant. Thus, using various parts of the spectrum, it is possible to give input data, or «instructions», to the plant which will lead to predictable biochemical events and tangible controlled practical results. In this case, the observed effects of controlling plant morphogenesis, based on the use of different lighting spectra, are in a sense related to gene modification, but do not change the plant’s gene pool itself [3].
The idea of using different spectral components of light to control plant development is not new. However, in order to understand why this approach is possible, it is necessary to know how plant responses are formed as a result of the expression of different genes under the influence of light. These studies are of great interest, since they open up the possibility of maximizing the genetic potential of plant crops without genetic modification or increased use of chemicals. The purpose of this article is to consider issues related to the light control of plant morphogenesis.
1.
PIGMENT PROTEINS IN PLANT ONTOGENESIS
In order for light to influence plant organisms and, in particular, to be used in the process of photosynthesis, it is necessary to absorb it with photosensitive proteins (antennas), i. e. pigments [4], which selectively absorb light. Th pigments play an important and diverse role in the life of organisms, especially in their photobiological processes.
The main photobiological process is photosynthesis, during which the energy of electromagnetic radiation is converted into the chemical energy of organic compounds [5].
The set, composition and ratio of pigments are specific for various groups of organisms [6]. Pigments of photosynthesis in higher plants are concentrated in plastids. They can be divided into four groups: chlorophylls, carotenoids, phycobilins, and flavonoids [7, 8].
Chlorophylls play a crucial role in the process of photosynthesis [9]. All higher plants contain chlorophylls a and b. Chlorophyll a in a solution has an absorption maximum at wavelengths of 440 and 700 nm, and chlorophyll b – at wavelengths of 460 and 660 nm. However, there are forms of chlorophyll that absorb light at a wavelength of 642, 710, and even 720 nm. The synthesis of chlorophyll is a multi-stage process proceeding with the participation of various enzymes, the formation of which is accelerated in the light. In the study of the effect of light on the formation of chlorophyll, in most cases the positive role of red light has been prominent. The intensity of lighting is also of great importance. There are lower and upper limits on the intensity of lighting of plants, starting from which the formation of chlorophyll is inhibited.
Along with green pigments, chloroplasts and chromatophores contain pigments belonging to the group of carotenoids. Carotenoids are yellow and orange pigments. They are present in all higher plants and in many microorganisms [10]. The main representatives of carotenoids in higher plants are two pigments – β-carotene (orange) and xanthophyll (yellow). β-carotene has two absorption maxima corresponding to wavelengths of 452 and 482 nm, and xanthophyll – at wavelengths of 470 and 502 nm. It was established that carotenoids, absorbing certain parts of the solar spectrum, transfer the energy of these rays to chlorophyll molecules and thereby contribute to the use of the spectral range of light that is not absorbed by chlorophyll. There is evidence that carotenoids also perform a protective function, protecting various organic substances of plant cells, primarily chlorophyll molecules, from destruction in the light during photooxidation. During leaf formation, carotenoids are formed and accumulate in plastids, and do not require light during synthesis.
Phycobilins are red and blue pigments present in cyanobacteria and some algae [11]. They are presented as the following pigments: phycocyanin, phycoerythrin and allophycocyanin. Phycobilins absorb rays in the green and yellow parts of the spectrum of light radiation. This is the part of the spectrum that lies between the two main absorption lines of chlorophyll. Phycoerythrin absorbs rays with a wavelength of 495–565 nm, and phycocyanin – at 550–615 nm. It is believed that phycobilins absorb the energy of light and, like carotenoids, transmit it to the chlorophyll molecule, after which it is used in photosynthesis.
Flavonoids are the largest class of plant pigments found in the form of glycosides in moisture of plants. These include anthocyanins, anthocyanidins, aurons, dihydrochalcones, isoflavones, catechins, leukoanthocyanidins, flavononols, flavones, flavanones, flavonols and chalcones. Depending on the pH of the medium, flavonoids have red, yellow, blue and violet color. They take part in photosynthesis, lignin formation, and are involved in the regulation of seed germination, proliferation, and cell death (by apoptosis) [12, 13].
It is known that the successful laying of generative structures and the ripening of fruits, seeds and other economically valuable organs of cultivated plants largely depend on the regulation of these processes, which involve numerous gene complexes. It is now clear that the size and stability of the antennas of the photosynthetic apparatus are important not only for the photosynthetic function, but also for the implementation of regulatory signals propagating beyond the chloroplasts of the plant cells [14].
During ontogenesis, plant cells should effectively coordinate the activity of two genomes – nuclear and plastid. Such coordination is possible due to the existence of two oppositely directed processes. On the one hand, there is a nuclear control over the expression of the genome of chloroplasts, on the other hand, there is reverse regulation directed from chloroplasts to the nucleus, which carries information on the state and functioning of these organelles under the specific conditions, and thus provides feedback between the cytoplasm and the nucleus. In this case, with a change in the spectral composition or light intensity, the stoichiometric composition of the proteins of the light-harvesting complexes of chloroplasts, as well as the intensity of the biosynthesis of chlorophylls, carotenoids, phycobilins and flavonoids, change. Almost all proteins involved in the implementation of these processes are encoded in the nucleus. In this regard, information on changes in the spectral composition or intensity of lighting should enter the nucleus from chloroplasts and lead to a change in the expression of the corresponding nuclear genes. The specific mechanisms of generation and transmission of plastid-nuclear signals in plants have not been studied to date. It is believed that such signals are reactive oxygen species generated with the participation of plastid-bound proteins, which, through a series of cascades with the participation of more stable forms of compounds, ensure the transmission of information through the cytoplasm to the nucleus [15, 16].
2.
PLANT PHOTOMORPHOGENESIS
Plant growth and development are controlled by genetic determinants, their expression products, and environmental signals. The physiological effects of light signals in plants are very differentiated: light is a unique source of energy that provides photosynthesis, but it also has a powerful stimulating effect on plant morphogenesis [1]. Photoreception is the most important function necessary for plants to adapt to lighting conditions and other environmental parameters, because light serves as a synchronizer of daily and seasonal biorhythms for them, as well as a source of specific signaling information [17]. Plant morphogenesis, which is controlled by lighting parameters, is called photomorphogenesis [18].
Today, several mechanisms of the regulatory influence of light on plants are considered generally accepted, the action of which can be both isolated or joint [16]:
the direct effect of light radiation on the genetic apparatus of plants through the excitation of photoreceptors, which contributes to the synthesis of necessary proteins;
endogenous regulation, manifested through the excitation by light of photoreceptors of the activity of phytohormones, which are one of the links of the regulatory system closest to photochromic proteins in plant cells;
the effect of light on the functional activity of cell membranes, carried out through a change in the electrical characteristics of the membranes of cells and tissues illuminated with light from plant organs, which causes certain physiological effects: the formation of phytohormones and the activation of certain genes.
Due to evolutionary adaptation to changing and extreme lighting conditions, plants have a sophisticated specialized photoreceptor network. In plants with the most developed light reception system responsible for the implementation of a variety of photoresponders, several types of regulatory photoreceptor proteins (photoreceptors) function, the spectral sensitivity of which allows for controlling morphogenesis of practically all areas of the optical spectrum. These include phytochromes, sensors of red (R) and far red (FR) light (operating range of 600–750 nm); cryptochroms and phototropins, receptors of near-ultraviolet (UV-A) and blue (B) light (operating range of 320–500 nm); as well as the UVR8 protein, a photon receptor in the far ultraviolet region of the spectrum (UV-B) (operating range of 290–320 nm) [19].
The expression of light-regulated genes in plants is controlled by various classes of photoreceptors [20,21], which transform light signals into biochemical signaling cascades that cause physiological cellular responses. The photon sensors of photoreceptor proteins are chromophore molecules, the photoconversion of which initiates structural changes in the photosensory domain, followed by signal transduction to the effector domains of the photoreceptors or interacting proteins, causing modulation of their activity.
Thus, the basis of plant photomorphogenesis is the detection by special photosensitive formations – photoreceptor proteins (photoreceptors) – of the presence or absence of light energy of a given intensity in a given wavelength range. It is assumed that the light signals received by photoreceptors must be converted and then transmitted through photoregulatory systems, causing gene expression, which ultimately leads to a physiological response. It has been proven that plant hormones, through the photoreception system, are also involved in the reaction to light. As a result, when predetermined changes in the parameters of light are detected, the photoreceptor starts a chain of biochemical processes that ultimately activate the desired reaction of the plant organism (Fig. 1).
The relationship between photoreceptor proteins and endogenous plant development programs is their effect on cell growth and development, which is manifested in the regulation of chloroplast movement, changes in membrane permeability, and the synthesis of enzymes and phytohormones. It is assumed that the absorbed quantum (or several quanta) of light converts the photoreceptor protein into an active form. Subsequently, a certain signal is generated that enters the cell nucleus to DNA, which derepresses the potentially active gene, bringing it into an active state, which results in switching in the matrix synthesis of messenger RNA (mRNA) and proteins.
Phytochrome genes are found in nuclear DNA. Therefore, gene expression is carried out in the nucleus, and phytochrome protein synthesis is carried out in cytoplasmic ribosomes. Phytochromobilin (phytochrome chromophore) is synthesized in plastids, and only then enters the cytoplasm. Autocatalytic covalent attachment of the chromophore to the phytochrome protein takes place in the cytoplasm. As a result, a functionally active phytochrome molecule is formed [22].
Thus, there is a two-way relationship between the signal systems and the plant genome: on the one hand, the proteins of the signal systems are encoded in the genome, and on the other, the signal systems control the genome, expressing or inhibiting the activity of other genes. Therefore, studies related to the study of plant signaling systems are intensively developing [3].
3.
PHYTOHORMONAL REGULATION OF PLANT ONTOGENESIS
The phytohormones synthesized in plant cells are low-molecular organic substances produced by plants and having regulatory functions. Phytohormones cause various physiological and morphological changes in parts of plants sensitive to their action. Substances traditionally considered phytohormones are auxins, gibberellins, cytokinins, ethylene, brassinosteroids and abscisic acid. Often, jasmonic and salicylic acids and some phenolic compounds are added to them [23,24].
Unlike animals, plants do not have special organs that synthesize hormones. However, some of their organs are more saturated with hormones compared to others. For example, apical stem meristems and the apical part of the root are enriched with auxins [25], abscisins usually act at the synthesis point, spreading only a short distance, and ethylene is transported only as a precursor [26].
Phytohormones have a wide spectrum of action and coordinate between individual cells and plant tissues. They regulate many processes of plant life: seed germination, growth, differentiation of tissues and organs, flowering and ripening of fruits. Forming in one organ (or its part) of a plant, phytohormones are usually transported to another organ (or its part). Exogenous phytohormones penetrate the plants quite evenly, and endogenous are localized in separate cell depots. Therefore, hormonal «feeding» of plants from the outside does not replace the natural synthesis of phytohormones and is able to help plants only under certain conditions, and therefore it is necessary to develop a controlled process for the synthesis of phytohormones.
The phytohormonal regulatory effect on plant growth and development is achieved in two ways: by changing the dose of phytohormone and the interaction of phytohormones. Depending on the concentration of phytohormone, its effect on the same process can vary from stimulation to inhibition. Furthermore, a change in its concentration can lead to a change in the nature of the action of the phytohormone and physiological response [27].
According to modern concepts, the regulatory effect of phytohormones is due to the fact that they regulate gene expression in a plant [28], and at the same time they act at different levels. Phytohormones interact in the plant cell with receptor proteins and form a kind of hormone-receptor complex, which then penetrates the nucleus and comes into contact with chromatin. Receptors are located both on membranes and in the cytosol. Therefore, the same hormone can bind to different receptors, thereby causing different physiological responses. This is one of the reasons for the multilevel action of phytohormones. At the first level, the direct interaction of phytohormone with DNA changes the structural state of chromatin and thereby affects its matrix activity. The second possible level of phytohormone effect is associated with the implementation of hereditary information through their influence on specific RNA polymerase enzymes that can «recognize» certain genes and synthesize giant molecules – precursors of messenger RNA (pre-mRNA). At the same time, phytohormones can regulate the life time of mRNA, as well as the process of its entry into the cytoplasm. Phytohormonal regulation of gene expression is possible at the level of translation – protein synthesis in ribosomes.
Thus, phytohormones not only regulate cell growth and development, but also are supra-cellular regulatory mechanisms. Methods of regulation can be different: some phytohormones can lower the expression of the target gene, others, on the contrary, can activate it. Therefore, competitive relationships arise between phytohormones. The formation and accumulation of one hormone instead of another, leads to a change in the nature of growth processes. Furthermore, one hormone can stimulate or inhibit the synthesis of another hormone.
Along with the differential effect on genome activity, the influence of phytohormones on the regulation of cell membrane permeability is of great importance. As a result of the association of the phytohormone with the membrane receptor, the membrane potential changes, which leads to the activation of the functional system of the cell, as a result of which the corresponding genes are activated / inactivated [26, 29, 30].
The hormonal system at each stage of plant development is characterized by a certain status: the state of the phytohormonal system in plant ontogenesis, the concentration level and the ratio between phytohormones in the processes of their formation, movement, use and inactivation in response to external influences [31]. Hormonal status can be changed by the influence of exogenous factors (various technological methods that change the growth conditions). Based on the above, it can be assumed that light is one of the significant factors in the change in the hormonal status of plants, and, consequently, in the regulation of their ontogenesis.
4.
EXPERIMENTAL RESULTS OF THE STUDY OF LIGHT CONTROL OF PLANT MORPHOGENESIS
A number of previous studies using monochromators and special spectral filters made it possible to study in detail the effect of the qualitative composition of light on plant development [32]. However, these studies still have not given an unambiguous answer to the question: how does light control the genome of plants and how to create an optimal lighting mode by combining the spectral components of radiation and their intensity to maximize the genetic potential of plants? Apparently, this was largely due to the absence of broadband radiation sources with a controlled spectral composition. One of the ways to obtain broadband light radiation is the joint use of various semiconductor LEDs in one lighting device. Modern LEDs cover an extremely wide range of radiation spectra – from ultraviolet to infrared [33]. Thus, combining a set of different LEDs, you can create multispectral controlled light sources that allow you to get light of any intensity and with almost any spectral composition. Fig. 2 shows the spectral characteristics of a controlled multi-element matrix LED light source described in [34, 35]. This light source allows for a given program to change the intensity, spectral composition and duration of the light flux.
The authors of [36] studies the influence of the spectral composition and intensity of broadband light radiation on the growth and development of plants using a multi-element matrix LED light source, the emission spectrum of which varied in the wavelength range of 440–660 nm and could be emitted both in mono and polychromatic modes, and close in spectral composition to the solar radiation spectrum (Fig. 2). The experiments were carried out on plants – regenerants of the potato Solanum tuberosum L., healed by the apical meristem method and cultivated in vitro. For control, plants grown under fluorescent lamps (LFW) were used. The experimental results illustrating the dynamics of plant development under the influence of different radiation spectra are presented in Fig. 3 [37].
The experiments showed that plants grown in blue light (B, RB) are low-growing, and in red (R, DR) – elongated. The leaves of the former are of normal size, the latter are underdeveloped and irregular in shape. These manifestations of ontogenesis are due to the fact that the red rays of the spectrum stimulate the cell stretching phase, and the blue-violet rays stimulate the differentiation phase. Plants grown under yellow and green radiation (Y, G) show elongation and poor development of foliage, i. e. monochromatic yellow and green light promotes rapid growth in length (brittle and thin plants are formed), but this does not lead to the accumulation of sufficient biomass. In general, for plants cultivated under monochromatic light (except for blue and deep blue), the maximum stem height was 1.5 times higher than that of the control group of plants grown under a fluorescent lamp. The greatest increase in stem length in these plant groups occurs due to the extension of internodes. The results obtained are apparently associated with the spectral dependence of monochromatic light stimulation of asthenia growth hormones: auxins and cytokinins.
The transition to polychromatic and broadband radiation (CW, W, WW, FS and SS) demonstrates a clear difference from monochromatic lighting, which manifests itself in a more intensive development of foliage and root system of plants. At the same time, the height of plants cultivated in polychromatic light is 0.6 times less than in control (fluorescent lamp – LFW). The greatest length and width of leaves are observed in plants grown in deep blue (RB) and cold white light (CW). The maximum mass of the aerial parts of plants was observed in specimens grown in polychromatic light (CW and W). This indicates the effectiveness of polychromatic light radiation having paired spectral maxima at wavelengths of 446.8 and 546.9 nm (CW), 446.8 and 550.2 nm (W), which stimulates the formation of chlorophylls and cratinoids, and affects the increase mass of plants.
Very often, versatility and effectiveness do not match. Studies on cenoses have shown that different types of plants exhibit different requirements for the optimal combination of spectral and energy characteristics of the light regime [38]. As noted above, the main role in the regulation of morphogenetic processes in plants is played by photoreceptors: phytochromes, cryptochromes and phototropin, which control plant cell phytohormones, the synthesis of which and exposure to light-activated photoreceptor proteins are either not studied at all or are not well understood. In particular, it is known that red and blue light changes the content of individual groups of phytohormones, which can manifest itself in the specificity of the action of the radiation spectrum on plant morphogenesis.
In vitro experiments were carried out on growing microclonal potato plants Solanum tuberosum L. when lighted with two-component radiation with wavelengths of 460 and 660 nm, which fall at the maximum absorption of chlorophyll b, as well as when lighted with broadband LED radiation (SS) in the wavelength range from 440 up to 660 nm. In the latter case, the SS radiation spectrum allows not only to excite chlorophyll a and b, but also to activate almost all plant photoreceptor proteins. Fig. 4 shows photographs of the dynamics of the development of microclonal plants in comparison with the control lighting with a white light fluorescent lamp. The experimental results showed that the use of SS radiation source allows to achieve an increase in plant biomass by 30% compared with two-wave irradiation and an increase in biomass by 17% in relation to control lighting. Thus, to ensure maximum plant productivity, it is advisable to use broadband light radiation.
The existing artificial light sources, with rare exceptions, cannot reproduce the solar spectrum in the range of photosynthetically active radiation or generate a similar spectrum, but with peak emissions at a number of separate frequencies (Fig. 1), which does not allow achieving their maximum efficiency. Therefore, the SS multicomponent matrix LED radiation source is of significant interest for increasing crop yields. At the same time, along with the selection of the spectral composition of the radiation, it is necessary to pay attention to the energy component of the radiation. In vitro studies with regenerated plants of S. tuberosum potatoes of the Rozhdestvensky and Bullfinch varieties, as well as of Stevia rebaudiana, showed the presence of an optimal intensity of broadband lighting of asthenia, at which their maximum response is achieved [39, 40].
Fig. 5 shows photographs illustrating the effectiveness of the development process of Stevia rebaudiana at different levels of its intensity of lighting by a broadband radiation source of the SS spectrum.
Perceiving light signals, photoreceptors initiate intracellular signaling pathways and thereby regulate plant development throughout the entire life cycle. A signal for starting plant morphogenesis is a change in the ratio of phytohormones of cytokinins and auxins, which are regulators of not only growth, but also differentiation of cellular structures. The interaction of plant hormones can be observed in the forms of synergism and antagonism. A synergistic effect is associated with a mutual increase in the action of hormones on any process. So, in particular, the regeneration of shoots from callus is activated by the action of cytokinins in the presence of auxins. As a rule, there are several ways in which callus cell development can go. Since photoreceptors play the main role in the regulation of morphogenetic processes by light, phytohormones are synthesized in the callus structure under the influence of light, which is activated by a different radiation spectrum through photoreceptor proteins.
In the culture of callus tissues, morphogenesis is understood as the emergence of organized structures from an unorganized mass of cells. In [34], the influence of the irradiation spectrum on the development of differentiated zones in the callus mass of the cell culture of rice of the Valley variety was studied. Fig. 6 shows photographs illustrating the week-wise dynamics of the development of callus culture in vitro.
It was found that for each radiation spectrum there is an optimal radiation intensity at which the maximum rate of development of the callus culture is observed. The table shows the results of observations of the temporary process of development of the callus cell culture of rice of the Valley variety under the influence of various radiation spectra, which allow us to trace the dynamics of the effect of the photoreceptor response regulated by various radiation spectra on the interactions of auxin and cytokinin hormones. It is known that auxins act on the growth of cells in two phases, depending on the concentration: they accelerate at low doses and inhibit growth processes at higher doses. In sterile tissue cultures, an increase in the content of cytokinins causes differentiation of cells depending on the concentration of the hormone.
In particular, under the action of the phytohormone cytokinin, chlorophyll is synthesized in plants. As can be seen, the presence of a significant part of blue light with a small red content in the polychromatic radiation of LFW and CW (the spectral composition of radiation is shown in Fig. 2) leads to an increase in the green (chlorophyll) mass, which is apparently due to an increase in the concentration of cytokinins with respect to auxins. A decrease or absence of the proportion of blue light in the emission spectrum inhibits the formation of chlorophyll mass, while an increase in the proportion of the red component leads to the formation of a brown (root) cell structure and an increase in callus mass, which is apparently due to an increase in the concentration of auxins.
Thus, the revealed process of the regulatory effect of the radiation spectrum on the key stages of callus cell differentiation, depending on the ratio of the hormones of auxins and cytokinins formed under the influence of the photoreceptor system in the cell mass of calluses, gives reason to talk about the need for further development of fundamental studies of the regulatory action of light, which should contribute to progress in creating new mechanisms of endogenous regulation of plant development.
CONCLUSION
Unlike animals, plants must initially respond to light in order to live. For this, they have a nature-created photoreceptor system. Therefore, light for plants is not only a source of energy, but also an important environmental factor that controls various signal transmission paths. Light is one of the main regulators of plant development and their metabolism. Gene expression in plants is regulated by light at many levels. The level of a gene product can be controlled by regulating the level of transcription of its gene or by regulating the translation of its mRNA into protein [41].
As of today, the relationship between the plant photoreceptor system and endogenous programs of their development remains poorly studied. Although light activation of the genetic apparatus of protein biosynthesis is no longer in doubt, it should be emphasized that, apparently, this effect is not the initial, but one of the final stages of the action of photoreceptor proteins, and the material nature of the signal propagating from the photoreceptor to the cell nucleus remains obscure. such reactions that occur almost immediately after lighting.
It is necessary to answer all these questions if humanity sets out to master the process of plant photomorphogenesis in order to increase the efficiency of crop production and the fullest disclosure of their genetic potential.
This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation, agreement number: 05.604.21.0229.
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Richard J. Mural Fundamentals of Light-Regulated Gene Expression // Plants Genetic Engineering. / Ed. Biswas B. B., Harris. J. R. Springer Nature Switzerland AG. 2019., p. 191–211.
Yu. N. Kulchin1,2, D. O. Goltsova1, E. P. Subbotin1
Institute of automation and control processes FEB RAS
Far Eastern Federal University, Vladivostok, Russia
The article deals with the issues of light regulation of the plant genetic system and light control of morphogenesis. The idea of the mechanisms of light signal translation in the cell is given. The relationship between photoreceptor proteins and endogenous programs of plant development is shown. The role of pigment proteins and phytohormones in the regulation of plant ontogenesis is characterized. Experimental results demonstrating light control of plant morphogenesis are presented.
Keywords: light, light control, spectrum, pigment proteins, photoreception, phytohormones, plants ontogenesis.
Received: 09.09.2019
Accepted: 07.10.2019
INTRODUCTION
Sunlight is an important adaptive stimulus, and many living organisms adapt their metabolism to the conditions of variable lighting in the environment, perceiving and reacting to light signals by changing their physiological functions [1]. In this case, light acts as a multifaceted factor characterized by qualitative (range of wavelengths) and quantitative (intensity, integral daily radiation, photoperiod) parameters, as well as direction and polarization. As a rule, the main reason for the low productivity of plants growing under natural conditions is that a huge amount of energy coming from the Sun is depreciated as a factor in photosynthesis due to unfavorable combinations of lighting parameters with other productivity conditions: heat, humidity, and soil fertility conditions [2]. Given that light controls the functioning of endogenous regulation systems (gene, enzymatic, trophic, hormonal, etc.), the combined effect of which provides an adequate response of plants to lighting conditions, by manipulating the characteristics of lighting, it is possible to maximize the potential determined by the genetic plan of the plant. Thus, using various parts of the spectrum, it is possible to give input data, or «instructions», to the plant which will lead to predictable biochemical events and tangible controlled practical results. In this case, the observed effects of controlling plant morphogenesis, based on the use of different lighting spectra, are in a sense related to gene modification, but do not change the plant’s gene pool itself [3].
The idea of using different spectral components of light to control plant development is not new. However, in order to understand why this approach is possible, it is necessary to know how plant responses are formed as a result of the expression of different genes under the influence of light. These studies are of great interest, since they open up the possibility of maximizing the genetic potential of plant crops without genetic modification or increased use of chemicals. The purpose of this article is to consider issues related to the light control of plant morphogenesis.
1.
PIGMENT PROTEINS IN PLANT ONTOGENESIS
In order for light to influence plant organisms and, in particular, to be used in the process of photosynthesis, it is necessary to absorb it with photosensitive proteins (antennas), i. e. pigments [4], which selectively absorb light. Th pigments play an important and diverse role in the life of organisms, especially in their photobiological processes.
The main photobiological process is photosynthesis, during which the energy of electromagnetic radiation is converted into the chemical energy of organic compounds [5].
The set, composition and ratio of pigments are specific for various groups of organisms [6]. Pigments of photosynthesis in higher plants are concentrated in plastids. They can be divided into four groups: chlorophylls, carotenoids, phycobilins, and flavonoids [7, 8].
Chlorophylls play a crucial role in the process of photosynthesis [9]. All higher plants contain chlorophylls a and b. Chlorophyll a in a solution has an absorption maximum at wavelengths of 440 and 700 nm, and chlorophyll b – at wavelengths of 460 and 660 nm. However, there are forms of chlorophyll that absorb light at a wavelength of 642, 710, and even 720 nm. The synthesis of chlorophyll is a multi-stage process proceeding with the participation of various enzymes, the formation of which is accelerated in the light. In the study of the effect of light on the formation of chlorophyll, in most cases the positive role of red light has been prominent. The intensity of lighting is also of great importance. There are lower and upper limits on the intensity of lighting of plants, starting from which the formation of chlorophyll is inhibited.
Along with green pigments, chloroplasts and chromatophores contain pigments belonging to the group of carotenoids. Carotenoids are yellow and orange pigments. They are present in all higher plants and in many microorganisms [10]. The main representatives of carotenoids in higher plants are two pigments – β-carotene (orange) and xanthophyll (yellow). β-carotene has two absorption maxima corresponding to wavelengths of 452 and 482 nm, and xanthophyll – at wavelengths of 470 and 502 nm. It was established that carotenoids, absorbing certain parts of the solar spectrum, transfer the energy of these rays to chlorophyll molecules and thereby contribute to the use of the spectral range of light that is not absorbed by chlorophyll. There is evidence that carotenoids also perform a protective function, protecting various organic substances of plant cells, primarily chlorophyll molecules, from destruction in the light during photooxidation. During leaf formation, carotenoids are formed and accumulate in plastids, and do not require light during synthesis.
Phycobilins are red and blue pigments present in cyanobacteria and some algae [11]. They are presented as the following pigments: phycocyanin, phycoerythrin and allophycocyanin. Phycobilins absorb rays in the green and yellow parts of the spectrum of light radiation. This is the part of the spectrum that lies between the two main absorption lines of chlorophyll. Phycoerythrin absorbs rays with a wavelength of 495–565 nm, and phycocyanin – at 550–615 nm. It is believed that phycobilins absorb the energy of light and, like carotenoids, transmit it to the chlorophyll molecule, after which it is used in photosynthesis.
Flavonoids are the largest class of plant pigments found in the form of glycosides in moisture of plants. These include anthocyanins, anthocyanidins, aurons, dihydrochalcones, isoflavones, catechins, leukoanthocyanidins, flavononols, flavones, flavanones, flavonols and chalcones. Depending on the pH of the medium, flavonoids have red, yellow, blue and violet color. They take part in photosynthesis, lignin formation, and are involved in the regulation of seed germination, proliferation, and cell death (by apoptosis) [12, 13].
It is known that the successful laying of generative structures and the ripening of fruits, seeds and other economically valuable organs of cultivated plants largely depend on the regulation of these processes, which involve numerous gene complexes. It is now clear that the size and stability of the antennas of the photosynthetic apparatus are important not only for the photosynthetic function, but also for the implementation of regulatory signals propagating beyond the chloroplasts of the plant cells [14].
During ontogenesis, plant cells should effectively coordinate the activity of two genomes – nuclear and plastid. Such coordination is possible due to the existence of two oppositely directed processes. On the one hand, there is a nuclear control over the expression of the genome of chloroplasts, on the other hand, there is reverse regulation directed from chloroplasts to the nucleus, which carries information on the state and functioning of these organelles under the specific conditions, and thus provides feedback between the cytoplasm and the nucleus. In this case, with a change in the spectral composition or light intensity, the stoichiometric composition of the proteins of the light-harvesting complexes of chloroplasts, as well as the intensity of the biosynthesis of chlorophylls, carotenoids, phycobilins and flavonoids, change. Almost all proteins involved in the implementation of these processes are encoded in the nucleus. In this regard, information on changes in the spectral composition or intensity of lighting should enter the nucleus from chloroplasts and lead to a change in the expression of the corresponding nuclear genes. The specific mechanisms of generation and transmission of plastid-nuclear signals in plants have not been studied to date. It is believed that such signals are reactive oxygen species generated with the participation of plastid-bound proteins, which, through a series of cascades with the participation of more stable forms of compounds, ensure the transmission of information through the cytoplasm to the nucleus [15, 16].
2.
PLANT PHOTOMORPHOGENESIS
Plant growth and development are controlled by genetic determinants, their expression products, and environmental signals. The physiological effects of light signals in plants are very differentiated: light is a unique source of energy that provides photosynthesis, but it also has a powerful stimulating effect on plant morphogenesis [1]. Photoreception is the most important function necessary for plants to adapt to lighting conditions and other environmental parameters, because light serves as a synchronizer of daily and seasonal biorhythms for them, as well as a source of specific signaling information [17]. Plant morphogenesis, which is controlled by lighting parameters, is called photomorphogenesis [18].
Today, several mechanisms of the regulatory influence of light on plants are considered generally accepted, the action of which can be both isolated or joint [16]:
the direct effect of light radiation on the genetic apparatus of plants through the excitation of photoreceptors, which contributes to the synthesis of necessary proteins;
endogenous regulation, manifested through the excitation by light of photoreceptors of the activity of phytohormones, which are one of the links of the regulatory system closest to photochromic proteins in plant cells;
the effect of light on the functional activity of cell membranes, carried out through a change in the electrical characteristics of the membranes of cells and tissues illuminated with light from plant organs, which causes certain physiological effects: the formation of phytohormones and the activation of certain genes.
Due to evolutionary adaptation to changing and extreme lighting conditions, plants have a sophisticated specialized photoreceptor network. In plants with the most developed light reception system responsible for the implementation of a variety of photoresponders, several types of regulatory photoreceptor proteins (photoreceptors) function, the spectral sensitivity of which allows for controlling morphogenesis of practically all areas of the optical spectrum. These include phytochromes, sensors of red (R) and far red (FR) light (operating range of 600–750 nm); cryptochroms and phototropins, receptors of near-ultraviolet (UV-A) and blue (B) light (operating range of 320–500 nm); as well as the UVR8 protein, a photon receptor in the far ultraviolet region of the spectrum (UV-B) (operating range of 290–320 nm) [19].
The expression of light-regulated genes in plants is controlled by various classes of photoreceptors [20,21], which transform light signals into biochemical signaling cascades that cause physiological cellular responses. The photon sensors of photoreceptor proteins are chromophore molecules, the photoconversion of which initiates structural changes in the photosensory domain, followed by signal transduction to the effector domains of the photoreceptors or interacting proteins, causing modulation of their activity.
Thus, the basis of plant photomorphogenesis is the detection by special photosensitive formations – photoreceptor proteins (photoreceptors) – of the presence or absence of light energy of a given intensity in a given wavelength range. It is assumed that the light signals received by photoreceptors must be converted and then transmitted through photoregulatory systems, causing gene expression, which ultimately leads to a physiological response. It has been proven that plant hormones, through the photoreception system, are also involved in the reaction to light. As a result, when predetermined changes in the parameters of light are detected, the photoreceptor starts a chain of biochemical processes that ultimately activate the desired reaction of the plant organism (Fig. 1).
The relationship between photoreceptor proteins and endogenous plant development programs is their effect on cell growth and development, which is manifested in the regulation of chloroplast movement, changes in membrane permeability, and the synthesis of enzymes and phytohormones. It is assumed that the absorbed quantum (or several quanta) of light converts the photoreceptor protein into an active form. Subsequently, a certain signal is generated that enters the cell nucleus to DNA, which derepresses the potentially active gene, bringing it into an active state, which results in switching in the matrix synthesis of messenger RNA (mRNA) and proteins.
Phytochrome genes are found in nuclear DNA. Therefore, gene expression is carried out in the nucleus, and phytochrome protein synthesis is carried out in cytoplasmic ribosomes. Phytochromobilin (phytochrome chromophore) is synthesized in plastids, and only then enters the cytoplasm. Autocatalytic covalent attachment of the chromophore to the phytochrome protein takes place in the cytoplasm. As a result, a functionally active phytochrome molecule is formed [22].
Thus, there is a two-way relationship between the signal systems and the plant genome: on the one hand, the proteins of the signal systems are encoded in the genome, and on the other, the signal systems control the genome, expressing or inhibiting the activity of other genes. Therefore, studies related to the study of plant signaling systems are intensively developing [3].
3.
PHYTOHORMONAL REGULATION OF PLANT ONTOGENESIS
The phytohormones synthesized in plant cells are low-molecular organic substances produced by plants and having regulatory functions. Phytohormones cause various physiological and morphological changes in parts of plants sensitive to their action. Substances traditionally considered phytohormones are auxins, gibberellins, cytokinins, ethylene, brassinosteroids and abscisic acid. Often, jasmonic and salicylic acids and some phenolic compounds are added to them [23,24].
Unlike animals, plants do not have special organs that synthesize hormones. However, some of their organs are more saturated with hormones compared to others. For example, apical stem meristems and the apical part of the root are enriched with auxins [25], abscisins usually act at the synthesis point, spreading only a short distance, and ethylene is transported only as a precursor [26].
Phytohormones have a wide spectrum of action and coordinate between individual cells and plant tissues. They regulate many processes of plant life: seed germination, growth, differentiation of tissues and organs, flowering and ripening of fruits. Forming in one organ (or its part) of a plant, phytohormones are usually transported to another organ (or its part). Exogenous phytohormones penetrate the plants quite evenly, and endogenous are localized in separate cell depots. Therefore, hormonal «feeding» of plants from the outside does not replace the natural synthesis of phytohormones and is able to help plants only under certain conditions, and therefore it is necessary to develop a controlled process for the synthesis of phytohormones.
The phytohormonal regulatory effect on plant growth and development is achieved in two ways: by changing the dose of phytohormone and the interaction of phytohormones. Depending on the concentration of phytohormone, its effect on the same process can vary from stimulation to inhibition. Furthermore, a change in its concentration can lead to a change in the nature of the action of the phytohormone and physiological response [27].
According to modern concepts, the regulatory effect of phytohormones is due to the fact that they regulate gene expression in a plant [28], and at the same time they act at different levels. Phytohormones interact in the plant cell with receptor proteins and form a kind of hormone-receptor complex, which then penetrates the nucleus and comes into contact with chromatin. Receptors are located both on membranes and in the cytosol. Therefore, the same hormone can bind to different receptors, thereby causing different physiological responses. This is one of the reasons for the multilevel action of phytohormones. At the first level, the direct interaction of phytohormone with DNA changes the structural state of chromatin and thereby affects its matrix activity. The second possible level of phytohormone effect is associated with the implementation of hereditary information through their influence on specific RNA polymerase enzymes that can «recognize» certain genes and synthesize giant molecules – precursors of messenger RNA (pre-mRNA). At the same time, phytohormones can regulate the life time of mRNA, as well as the process of its entry into the cytoplasm. Phytohormonal regulation of gene expression is possible at the level of translation – protein synthesis in ribosomes.
Thus, phytohormones not only regulate cell growth and development, but also are supra-cellular regulatory mechanisms. Methods of regulation can be different: some phytohormones can lower the expression of the target gene, others, on the contrary, can activate it. Therefore, competitive relationships arise between phytohormones. The formation and accumulation of one hormone instead of another, leads to a change in the nature of growth processes. Furthermore, one hormone can stimulate or inhibit the synthesis of another hormone.
Along with the differential effect on genome activity, the influence of phytohormones on the regulation of cell membrane permeability is of great importance. As a result of the association of the phytohormone with the membrane receptor, the membrane potential changes, which leads to the activation of the functional system of the cell, as a result of which the corresponding genes are activated / inactivated [26, 29, 30].
The hormonal system at each stage of plant development is characterized by a certain status: the state of the phytohormonal system in plant ontogenesis, the concentration level and the ratio between phytohormones in the processes of their formation, movement, use and inactivation in response to external influences [31]. Hormonal status can be changed by the influence of exogenous factors (various technological methods that change the growth conditions). Based on the above, it can be assumed that light is one of the significant factors in the change in the hormonal status of plants, and, consequently, in the regulation of their ontogenesis.
4.
EXPERIMENTAL RESULTS OF THE STUDY OF LIGHT CONTROL OF PLANT MORPHOGENESIS
A number of previous studies using monochromators and special spectral filters made it possible to study in detail the effect of the qualitative composition of light on plant development [32]. However, these studies still have not given an unambiguous answer to the question: how does light control the genome of plants and how to create an optimal lighting mode by combining the spectral components of radiation and their intensity to maximize the genetic potential of plants? Apparently, this was largely due to the absence of broadband radiation sources with a controlled spectral composition. One of the ways to obtain broadband light radiation is the joint use of various semiconductor LEDs in one lighting device. Modern LEDs cover an extremely wide range of radiation spectra – from ultraviolet to infrared [33]. Thus, combining a set of different LEDs, you can create multispectral controlled light sources that allow you to get light of any intensity and with almost any spectral composition. Fig. 2 shows the spectral characteristics of a controlled multi-element matrix LED light source described in [34, 35]. This light source allows for a given program to change the intensity, spectral composition and duration of the light flux.
The authors of [36] studies the influence of the spectral composition and intensity of broadband light radiation on the growth and development of plants using a multi-element matrix LED light source, the emission spectrum of which varied in the wavelength range of 440–660 nm and could be emitted both in mono and polychromatic modes, and close in spectral composition to the solar radiation spectrum (Fig. 2). The experiments were carried out on plants – regenerants of the potato Solanum tuberosum L., healed by the apical meristem method and cultivated in vitro. For control, plants grown under fluorescent lamps (LFW) were used. The experimental results illustrating the dynamics of plant development under the influence of different radiation spectra are presented in Fig. 3 [37].
The experiments showed that plants grown in blue light (B, RB) are low-growing, and in red (R, DR) – elongated. The leaves of the former are of normal size, the latter are underdeveloped and irregular in shape. These manifestations of ontogenesis are due to the fact that the red rays of the spectrum stimulate the cell stretching phase, and the blue-violet rays stimulate the differentiation phase. Plants grown under yellow and green radiation (Y, G) show elongation and poor development of foliage, i. e. monochromatic yellow and green light promotes rapid growth in length (brittle and thin plants are formed), but this does not lead to the accumulation of sufficient biomass. In general, for plants cultivated under monochromatic light (except for blue and deep blue), the maximum stem height was 1.5 times higher than that of the control group of plants grown under a fluorescent lamp. The greatest increase in stem length in these plant groups occurs due to the extension of internodes. The results obtained are apparently associated with the spectral dependence of monochromatic light stimulation of asthenia growth hormones: auxins and cytokinins.
The transition to polychromatic and broadband radiation (CW, W, WW, FS and SS) demonstrates a clear difference from monochromatic lighting, which manifests itself in a more intensive development of foliage and root system of plants. At the same time, the height of plants cultivated in polychromatic light is 0.6 times less than in control (fluorescent lamp – LFW). The greatest length and width of leaves are observed in plants grown in deep blue (RB) and cold white light (CW). The maximum mass of the aerial parts of plants was observed in specimens grown in polychromatic light (CW and W). This indicates the effectiveness of polychromatic light radiation having paired spectral maxima at wavelengths of 446.8 and 546.9 nm (CW), 446.8 and 550.2 nm (W), which stimulates the formation of chlorophylls and cratinoids, and affects the increase mass of plants.
Very often, versatility and effectiveness do not match. Studies on cenoses have shown that different types of plants exhibit different requirements for the optimal combination of spectral and energy characteristics of the light regime [38]. As noted above, the main role in the regulation of morphogenetic processes in plants is played by photoreceptors: phytochromes, cryptochromes and phototropin, which control plant cell phytohormones, the synthesis of which and exposure to light-activated photoreceptor proteins are either not studied at all or are not well understood. In particular, it is known that red and blue light changes the content of individual groups of phytohormones, which can manifest itself in the specificity of the action of the radiation spectrum on plant morphogenesis.
In vitro experiments were carried out on growing microclonal potato plants Solanum tuberosum L. when lighted with two-component radiation with wavelengths of 460 and 660 nm, which fall at the maximum absorption of chlorophyll b, as well as when lighted with broadband LED radiation (SS) in the wavelength range from 440 up to 660 nm. In the latter case, the SS radiation spectrum allows not only to excite chlorophyll a and b, but also to activate almost all plant photoreceptor proteins. Fig. 4 shows photographs of the dynamics of the development of microclonal plants in comparison with the control lighting with a white light fluorescent lamp. The experimental results showed that the use of SS radiation source allows to achieve an increase in plant biomass by 30% compared with two-wave irradiation and an increase in biomass by 17% in relation to control lighting. Thus, to ensure maximum plant productivity, it is advisable to use broadband light radiation.
The existing artificial light sources, with rare exceptions, cannot reproduce the solar spectrum in the range of photosynthetically active radiation or generate a similar spectrum, but with peak emissions at a number of separate frequencies (Fig. 1), which does not allow achieving their maximum efficiency. Therefore, the SS multicomponent matrix LED radiation source is of significant interest for increasing crop yields. At the same time, along with the selection of the spectral composition of the radiation, it is necessary to pay attention to the energy component of the radiation. In vitro studies with regenerated plants of S. tuberosum potatoes of the Rozhdestvensky and Bullfinch varieties, as well as of Stevia rebaudiana, showed the presence of an optimal intensity of broadband lighting of asthenia, at which their maximum response is achieved [39, 40].
Fig. 5 shows photographs illustrating the effectiveness of the development process of Stevia rebaudiana at different levels of its intensity of lighting by a broadband radiation source of the SS spectrum.
Perceiving light signals, photoreceptors initiate intracellular signaling pathways and thereby regulate plant development throughout the entire life cycle. A signal for starting plant morphogenesis is a change in the ratio of phytohormones of cytokinins and auxins, which are regulators of not only growth, but also differentiation of cellular structures. The interaction of plant hormones can be observed in the forms of synergism and antagonism. A synergistic effect is associated with a mutual increase in the action of hormones on any process. So, in particular, the regeneration of shoots from callus is activated by the action of cytokinins in the presence of auxins. As a rule, there are several ways in which callus cell development can go. Since photoreceptors play the main role in the regulation of morphogenetic processes by light, phytohormones are synthesized in the callus structure under the influence of light, which is activated by a different radiation spectrum through photoreceptor proteins.
In the culture of callus tissues, morphogenesis is understood as the emergence of organized structures from an unorganized mass of cells. In [34], the influence of the irradiation spectrum on the development of differentiated zones in the callus mass of the cell culture of rice of the Valley variety was studied. Fig. 6 shows photographs illustrating the week-wise dynamics of the development of callus culture in vitro.
It was found that for each radiation spectrum there is an optimal radiation intensity at which the maximum rate of development of the callus culture is observed. The table shows the results of observations of the temporary process of development of the callus cell culture of rice of the Valley variety under the influence of various radiation spectra, which allow us to trace the dynamics of the effect of the photoreceptor response regulated by various radiation spectra on the interactions of auxin and cytokinin hormones. It is known that auxins act on the growth of cells in two phases, depending on the concentration: they accelerate at low doses and inhibit growth processes at higher doses. In sterile tissue cultures, an increase in the content of cytokinins causes differentiation of cells depending on the concentration of the hormone.
In particular, under the action of the phytohormone cytokinin, chlorophyll is synthesized in plants. As can be seen, the presence of a significant part of blue light with a small red content in the polychromatic radiation of LFW and CW (the spectral composition of radiation is shown in Fig. 2) leads to an increase in the green (chlorophyll) mass, which is apparently due to an increase in the concentration of cytokinins with respect to auxins. A decrease or absence of the proportion of blue light in the emission spectrum inhibits the formation of chlorophyll mass, while an increase in the proportion of the red component leads to the formation of a brown (root) cell structure and an increase in callus mass, which is apparently due to an increase in the concentration of auxins.
Thus, the revealed process of the regulatory effect of the radiation spectrum on the key stages of callus cell differentiation, depending on the ratio of the hormones of auxins and cytokinins formed under the influence of the photoreceptor system in the cell mass of calluses, gives reason to talk about the need for further development of fundamental studies of the regulatory action of light, which should contribute to progress in creating new mechanisms of endogenous regulation of plant development.
CONCLUSION
Unlike animals, plants must initially respond to light in order to live. For this, they have a nature-created photoreceptor system. Therefore, light for plants is not only a source of energy, but also an important environmental factor that controls various signal transmission paths. Light is one of the main regulators of plant development and their metabolism. Gene expression in plants is regulated by light at many levels. The level of a gene product can be controlled by regulating the level of transcription of its gene or by regulating the translation of its mRNA into protein [41].
As of today, the relationship between the plant photoreceptor system and endogenous programs of their development remains poorly studied. Although light activation of the genetic apparatus of protein biosynthesis is no longer in doubt, it should be emphasized that, apparently, this effect is not the initial, but one of the final stages of the action of photoreceptor proteins, and the material nature of the signal propagating from the photoreceptor to the cell nucleus remains obscure. such reactions that occur almost immediately after lighting.
It is necessary to answer all these questions if humanity sets out to master the process of plant photomorphogenesis in order to increase the efficiency of crop production and the fullest disclosure of their genetic potential.
This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation, agreement number: 05.604.21.0229.
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