Changes in Photosynthetic Pigments Content in Non-Transformed and AtCKX Transgenic Centaury ( Centaurium erythraea Rafn) Shoots Grown under Salt Stress In Vitro

: The effects of graded sodium chloride (NaCl) concentrations (0-, 50-, 100-, 150-, and 200-mM) on photosynthetic pigment contents in in vitro grown shoots of important medicinal plant species ( Centaurium erythraea Rafn) were investigated. Non-transformed, one AtCKX1 and two AtCKX2 transgenic centaury lines, with altered cytokinin proﬁles, were used in this study. The chlorophyll ( Chl ) and carotenoid contents differed in the non-transformed and transgenic lines. In general, salinity signiﬁcantly reduced the Chl a and Chl b contents in comparison to the NaCl-free medium. The lowest Chl content was observed in AtCKX2 transgenic shoots grown on all the culture media. The total carotenoid content was increased in shoots of non-transformed and both AtCKX2 transgenic lines grown in 50-mM NaCl. On the other hand, in concentrations >50-mM NaCl, the total carotenoid content was decreased in all analysed centaury shoots. The Chl a / Chl b ratio in all the shoots increased progressively in the graded NaCl concentrations. Contrarily, the addition of NaCl in the culture medium reduced the Chl /carotenoid ratio in centaury shoots. Taken together, the results of this study partly explained the mode of centaury plant adaptations to salt stress in vitro. Thus, the results on centaury shoots conﬁrmed that the determination of the photosynthetic pigment contents can be a very useful non-destructive screening method in order to discriminate susceptible and resistant plant species/lines to salt stress conditions.


Introduction
Common centaury (Centaurium erythraea Rafn) belongs to the Gentianaceae family and represents an important medicinal plant species distributed throughout the Northern Hemisphere. In traditional medicine, centaury has for centuries been used to cure numerous health issues such as febrile conditions, blood sugar, anaemia, jaundice, gout, etc. [1]. Centaury is a cosmopolitan plant species that inhabits mountain slopes, dry grasslands, and scrublands. In addition, centaury can also be found in saline soils.
Salinity is one of the most significant environmental stresses limiting cell metabolism and plant productivity [2]. The detrimental salinity effects on plants are the consequence of Na + and Cl − ion toxicity and, also, a water deficit that results in osmotic and ionic stress, which usually leads to numerous physiological and biochemical changes that inhibit plant growth, development, and protein synthesis [3]. Salinity stress induces remarkable alternations in physiological, biochemical, and molecular processes [4]. Photosynthesis is an important parameter used to monitor plant responses to abiotic stress. Reduced photosynthesis under salinity is not only attributed to stomata closure and reduced intercellular roots grown in vitro. It was shown that centaury roots showed a higher salinity tolerance compared to shoots. Considering the fact that maintaining the Chl content contributes to a salt tolerance, in this study, we investigated the influence of salinity in vitro on the photosynthetic pigments of non-transformed and AtCKX transgenic centaury shoots with a reduced amount of bioactive CKs.

Plant Material, Growth Media, and Culture Conditions
Non-transformed and transgenic AtCKX centaury plants were obtained as previously described [38]. One AtCKX1 (line 29) and two AtCKX2 (lines 17 and 26) transgenic centaury lines were selected as the starting plant materials. The selection of these three transgenic lines was based on the previously described overexpression of AtCKX genes in centaury shoots and roots, which resulted in an altered CKs profile, leading to a decline of the bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms, and/or CK nucleotides [32]. The shoots of non-transformed and transgenic AtCKX lines were grown in vitro on half-strength plant growth regulator-free MS medium ( 1 /2MS [39]) solidified with 0.7% agar (Torlak, Belgrade, Serbia) and supplemented with 3% sucrose and 100-mg L −1 myo-inositol (Sigma-Aldrich, St. Louis, MO, USA). Four weeks after the last subculture, isolated centaury shoots of the non-transformed and all the transgenic AtCKX lines were grown on new 1 /2MS plant growth regulator-free medium or 1 /2MS medium supplemented with a range of NaCl (Lach-Ner, Neratovice, Czech Republic) concentrations (50-, 100-, 150-, and 200-mM) during the next eight weeks. The medium was adjusted to pH 5.8 with NaOH/HCl and autoclaved at 121 • C for 25 min. All in vitro cultured plants were grown at 25 ± 2 • C and at a 16-h/8-h photoperiod ('Tesla' white fluorescent lamps, 65 W, 4500 K; light flux of 47 µmols −1 m −2 ). The experiments were repeated three times, with 90 explants per each treatment.

Quantification of Photosynthetic Pigments
The isolation of the Chl and carotenoids was accomplished from the leaves of nontransformed and AtCKX transgenic centaury shoots after eight weeks of cultivation. Pigment extraction was done from leaves collected in the bottom part of the rosette. The total Chl and carotenoid contents were extracted using 96% ethanol. The plant material was heated in a water bath at 70 • C. The absorbance of the pigments was further measured with a UV-visible spectrophotometer (Agilent 8453, Life Sciences, Santa Clara, CA, USA) at 470, 648, and 664 nm. The concentrations of Chl a, Chl b, and the carotenoids were calculated using the equations proposed by Lichtenthaler [40]: The Chl a/Chl b ratio was calculated from the quotient of Chl a and Chl b (C a /C b ), while the Chl/carotenoids ratio was calculated from the quotient of their total values (C (a+b) /C (x+c) ). Determination of the photosynthetic pigments was accomplished in three biological samples per those non-transformed and each of the transgenic lines. Additionally, the absorbance of the supernatant was measured three times for each sample. The concentrations of all the analysed photosynthetic pigments were presented as µg/g of fresh sample weight (FW).

Statistical Analysis
The effect of a range of NaCl concentrations (0-200 mM) on the photosynthetic pigment contents of 8-week-old non-transformed and transgenic AtCKX shoots were evaluated using a standard two-factor analysis of variance (ANOVA). The results were presented as the mean ± SE. The comparisons between the mean values were made using Fisher's LSD (least significant difference) post-hoc test calculated at a confidence level of p ≤ 0.05.

Effect of NaCl on Photosynthetic Pigments Content of Centaury Shoots Grown In Vitro
The shoots of non-transformed and three transgenic AtCKX lines successfully survived eight weeks on 1 /2MS medium supplemented with a range of NaCl concentrations (0-, 50-, 100-, 150-, and 200-mM). The morphological appearance of the shoots in the non-transformed and AtCKX lines grown on NaCl-free medium showed normal rosette morphology and oval leaves with dark green colour. However, salt stress deteriorated the phenotypic appearances in all the salt-treated centaury shoots ( Figure 1). Cultivation of centaury shoots in increased salt-enriched mediums caused the rolling and burning of the leaf tips and yellowing of the whole plant. With an increase of the NaCl concentrations in the culture media, the profiles of the leaves evolved from green to lighter tones. In addition, most yellow leaves with altered morphology were observed on shoots grown in culture media supplemented with 150-and 200-mM NaCl. The chlorosis of leaves is one of the most common symptoms of salt stress in plants due to the decreasing amounts of photosynthetic pigments. In the common centaury cultures, chlorosis reached higher levels with the increased NaCl concentrations. The highest level of chlorosis at the highest salinity rate (200-mM) in both the non-transformed and transgenic lines was observed. Visible leaf damage was detected in all the centaury lines, but the leaves of the ATCKX2-26 line exhibited drastic senescence symptoms, as indicated by the intense necrotic areas in the leaves ( Figure 1). Moreover, it was interesting to note that the non-transformed and all the AtCKX transgenic centaury shoots spontaneously rooted in NaCl-free and the medium supplemented only with 50-mM NaCl after eight weeks. The total Chl (Chl a + Chl b) content is shown in Figure 2a. It was evident that the biosynthesis of Chl in common centaury was altered and affected by the salinity stress. When the shoots were grown in NaCl-free medium, significant differences in the total Chl content between the non-transformed and transgenic AtCKX lines were observed. In the  The total Chl (Chl a + Chl b) content is shown in Figure 2a. It was evident that the biosynthesis of Chl in common centaury was altered and affected by the salinity stress. When the shoots were grown in NaCl-free medium, significant differences in the total Chl content between the non-transformed and transgenic AtCKX lines were observed. In the non-transformed and transgenic line AtCKX1-29, the total Chl content was similar in the shoots grown in NaCl-free medium, while the total Chl contents in both AtCKX2 transgenic lines varied greatly. One transgenic line AtCKX2-26 had a significantly higher total Chl content in comparison to the non-transformed shoots and transgenic line AtCKX1-29. A higher total Chl content in this transgenic line was the consequence of a higher content of Chl b, which was detected in this line in comparison to all the other shoots ( Figure 2c). Contrarily, the other AtCKX2 transgenic line (17) showed a several times lower total Chl content when the shoots were grown in the NaCl-free medium. Regarding the response to salinity, all the analysed centaury shoots showed different patterns. In the non-transformed shoots and transgenic lines AtCKX1-29 and AtCKX2-26, a decreased total Chl content started with a slight salinity application (50-mM NaCl). Contrarily, in transgenic line AtCKX2-17, a significant decrement of the total Chl content was observed in shoots grown in a two-times higher NaCl concentration (100-mM). Although the same trend of Chl degradation was noticed in all the analysed centaury lines, significant differences were detected. The total Chl content was decreased in all the AtCKX transgenic lines in 150-and 200-mM NaCl in comparison to the non-transformed shoots. The effect of salinity on Chl a and Chl b was separately analysed (Figure 2b,c). Different patterns in the decrement of Chl a in the non-transformed and transgenic ACKX shoots were observed. A rapid decrement in the Chl a content was observed at concentrations >100-mM in the shoots of non-transformed and both AtCKX2 transgenic lines, while, in  The effect of salinity on Chl a and Chl b was separately analysed (Figure 2b,c). Different patterns in the decrement of Chl a in the non-transformed and transgenic ACKX shoots were observed. A rapid decrement in the Chl a content was observed at concentrations >100-mM in the shoots of non-transformed and both AtCKX2 transgenic lines, while, in the shoots of the AtCKX1-29 transgenic line, a significant decrement of the Chl a content was detected even in 50-mM NaCl. Regardless of the initial values, the Chl a content continued to decrease until 200-mM NaCl in all the analysed centaury shoots. It was interesting to note that, as in the case of the total Chl content, the Chl a content was decreased in all the AtCKX transgenic lines in 150-and 200-mM NaCl in comparison to the non-transformed shoots. The alteration of the Chl b content was also noticed in centaury shoots under salinity conditions. When the NaCl concentration in the medium increased, the Ch b content in the shoots was accordingly decreased. The content of Chl b in the shoots of the non-transformed and transgenic lines greatly varied. In the non-transformed and AtCKX2-26 transgenic line, the similar Chl b contents in the shoots grown at 50-mM of NaCl were determined, while, in the other concentrations, a significantly rapid decrement was observed. The smallest Chl b content was detected in 200-mM NaCl in all the centaury shoots.
The total carotenoid content was evaluated in all the centaury shoots ( Figure 3). It is evident that the total carotenoid content was different in the transgenic lines in comparison to the non-transgenic line when they were grown in the NaCl medium. When the non-transformed and AtCKX2 transgenic lines were grown in NaCl-free medium, a similar total carotenoid content was observed, while, in the AtCKX1 transgenic line, the carotenoid content was significantly lower. Additionally, the same pattern was recorded in slightly saline conditions. In 50-mM NaCl, the total carotenoid content was increased in the non-transformed shoots and AtCKX2 transgenic lines in comparison to the NaClfree medium. In the AtCKX1 transgenic shoots, the total carotenoid content remained unchanged. Further, in 100-mM NaCl, the total carotenoid content continued to decrease slowly in the non-transformed and AtCKX1 shoots, while, in the AtCKX2 transgenic shoots, a more rapid decrease in the total carotenoid content was observed. In comparison to the non-transformed shoots, in all the AtCKX transgenic lines, the most decreased total carotenoid content was detected in 150-and 200-mM NaCl. comparison to the non-transgenic line when they were grown in the NaCl medium. When the non-transformed and AtCKX2 transgenic lines were grown in NaCl-free medium, a similar total carotenoid content was observed, while, in the AtCKX1 transgenic line, the carotenoid content was significantly lower. Additionally, the same pattern was recorded in slightly saline conditions. In 50-mM NaCl, the total carotenoid content was increased in the non-transformed shoots and AtCKX2 transgenic lines in comparison to the NaClfree medium. In the AtCKX1 transgenic shoots, the total carotenoid content remained unchanged. Further, in 100-mM NaCl, the total carotenoid content continued to decrease slowly in the non-transformed and AtCKX1 shoots, while, in the AtCKX2 transgenic shoots, a more rapid decrease in the total carotenoid content was observed. In comparison to the non-transformed shoots, in all the AtCKX transgenic lines, the most decreased total carotenoid content was detected in 150-and 200-mM NaCl.

Effect of NaCl on Photosynthetic Pigments Ratio
The evaluation of the Chl a/Chl b ratio of the common century non-transformed and AtCKX transgenic lines is shown in Figure 4. The non-transformed and transgenic lines showed different Chl a/Chl b ratios in the shoots grown, even those grown in the NaClfree medium. The non-transformed and two transgenic centaury lines (AtCKX1-29 and AtCKX2-26) showed similar Chl a/Chl b ratios, while, in transgenic line AtCKX2-17, an almost doubled Chl a/Chl b ratio was noticed when the shoots were grown in NaCl-free medium. Regardless of the NaCl, an incremental increase in the nutrition medium in the Chl a/Chl b ratios in all the analysed centaury lines was observed. In shoots grown in 200-

Effect of NaCl on Photosynthetic Pigments Ratio
The evaluation of the Chl a/Chl b ratio of the common century non-transformed and AtCKX transgenic lines is shown in Figure 4. The non-transformed and transgenic lines showed different Chl a/Chl b ratios in the shoots grown, even those grown in the NaClfree medium. The non-transformed and two transgenic centaury lines (AtCKX1-29 and AtCKX2-26) showed similar Chl a/Chl b ratios, while, in transgenic line AtCKX2-17, an almost doubled Chl a/Chl b ratio was noticed when the shoots were grown in NaCl-free medium. Regardless of the NaCl, an incremental increase in the nutrition medium in the Chl a/Chl b ratios in all the analysed centaury lines was observed. In shoots grown in 200-mM NaCl, the highest Chl a/Chl b ratio was determined in the non-transformed and AtCKX1-17 and AtCKX2-29 transgening lines. The lowest change in the Chl a/Chl b ratio under salinity stress was achieved in the AtCKX2-26 transgenic line.  The ratio of the total Chl and total carotenoids determines the level of plant senescence. The obtained results in our study showed a different total Chl/carotenoid ratio in the non-transformed and transgenic lines ( Figure 5). As in the case of the Chl a/Chl b ratio, when the non-transformed and two transgenic centaury lines (AtCKX1-29 and AtCKX2-26) are grown in a NaCl-free medium, a similar total Chl/total carotenoids ratio was detected. In the same medium, transgenic line AtCKX2-17 showed about a three times lower initial total Chl/total carotenoids ratio than the non-transformed and other transgenic lines. The addition, the 50-mM NaCl medium remarkably reduced the total Chl/total carotenoids ratio in the non-transformed (14.84 → 7.61), AtCKX1-29 (18.05 → 3.56), and AtCKX2-26 (16.49 → 6.66) transgenic shoots, while, in the shoots of AtCKX2-17, the reduction was lower (5.59 → 3.94). It is obvious that a significant reduction of the Chl/total carotenoids ratio was detected in the transgenic centaury lines with regards to the nontransformed line. The reduction of the Chl/total carotenoid ratio was further continued in 100-mM NaCl and maintained at a similar level in 150-and 200-mM NaCl in all the centaury lines. The ratio of the total Chl and total carotenoids determines the level of plant senescence. The obtained results in our study showed a different total Chl/carotenoid ratio in the non-transformed and transgenic lines ( Figure 5). As in the case of the Chl a/Chl b ratio, when the non-transformed and two transgenic centaury lines (AtCKX1-29 and AtCKX2-26) are grown in a NaCl-free medium, a similar total Chl/total carotenoids ratio was detected. In the same medium, transgenic line AtCKX2-17 showed about a three times lower initial total Chl/total carotenoids ratio than the non-transformed and other transgenic lines. The addition, the 50-mM NaCl medium remarkably reduced the total Chl/total carotenoids ratio in the non-transformed (14.84 → 7.61), AtCKX1-29 (18.05 → 3.56), and AtCKX2-26 (16.49 → 6.66) transgenic shoots, while, in the shoots of AtCKX2-17, the reduction was lower (5.59 → 3.94). It is obvious that a significant reduction of the Chl/total carotenoids ratio was detected in the transgenic centaury lines with regards to the non-transformed line. The reduction of the Chl/total carotenoid ratio was further continued in 100-mM NaCl and maintained at a similar level in 150-and 200-mM NaCl in all the centaury lines.
rotenoids ratio in the non-transformed (14.84 → 7.61), AtCKX1-29 (18.05 → 3.56), and AtCKX2-26 (16.49 → 6.66) transgenic shoots, while, in the shoots of AtCKX2-17, the reduction was lower (5.59 → 3.94). It is obvious that a significant reduction of the Chl/total carotenoids ratio was detected in the transgenic centaury lines with regards to the nontransformed line. The reduction of the Chl/total carotenoid ratio was further continued in 100-mM NaCl and maintained at a similar level in 150-and 200-mM NaCl in all the centaury lines.

Discussion
In this study, we postulated that a reduced amount of bioactive CKs in AtCKX transgenic centaury plants could alter the photosynthetic pigment contents in shoots grown under salt stress in vitro. There is a lot of evidence in the literature that the phenomenon of senescence is also regulated by CKs [41] and reducing the Chl content represents one of the significant senescence markers [42][43][44][45]. It is also known that salt stress causes progressive Chl degradation, which leads to a reduced light absorption in plant leaves [5,[46][47][48]. The results of this work showed that, in the transgenic AtCKX2-17 shoots, an almost three times lower total Chl content was observed in comparison to the non-transformed shoots. Thus, it can be assumed that a reduced amount of cytokinin speeds up senescence. On the other hand, a total Chl content similar to that in the non-transformed shoots was determined in the transgenic line AtCKX1-29, while a significantly higher total Chl content was observed in AtCKX2-26. An increased total Chl content was also obtained in the transgenic AtCKX2 tobacco line, where the leaves remained green longer than the control leaves, which turned yellow faster. In addition, it was observed that the amount of total Chl in the transgenic tobacco leaves decreased during the time [49]. Despite our theoretical expectations, the transgenic AtCKX leaves of Arabidopsis thaliana also did not show accelerated senescence [28]. Some previous investigations also showed that an increased CKX activity in senescing barley [50] and a high expression of the CKX genes throughout the senescence in Trifolium repens [51] support our findings that CKX genes in fact do not induce senescence but facilitate the progression of senescence. Ciobanu and Sumalan [52] reported that the tomato Chl content decreased with an increasing salinity, and the changes in the Chl content of the leaves occurred barely after a prolonged growth of six weeks. The modulation of the photosynthetic performance in Pennisetum leaves under long-term moderate salinity stress also involved the inhibition of leaf pigment synthesis [53].
Soil salinity and saline habits, as well as arid climates, greatly affect the synthesis of plant pigment components [54]. In general, the decrement of photosynthetic pigment contents under salt stress is considered to be a result of the slow synthesis or fast breakdown of pigments [55]. In all the centaury shoots, decreased Chl a and Chl b, as well as the total Chl content in increased NaCl concentrations in the culture medium, were recorded. These results are in accordance with a previous work, which reported that salt stress induced with NaCl caused a decrement in the total Chl content in Phaseolus vulgaris [56], Catharanthus roseus [57], Vigna unguiculata [58], Vigna subterranean [59], Ocimum basilicum [60], and Solanum lycopersicum [61]. Recent research showed that salinity significantly decreased the Chl content and photosynthetic efficiency of sweet sorghum [62] and peppermint plants [63]. It was also reported that Chl a, Chl b, and the total Chl content in centaury leaves decreased with the increment of the salt concentration [36]. The reduction of Chl under salt stress could be attributed to the increased activity of photosynthetic enzymes and instability of the pigment-protein complex [5,[64][65][66]. Khan [67] also reported that saline stress slowed the production of the photosynthetic pigments. The reduction of the total carotenoid content was observed in all the centaury shoots cultured in mediums supplemented with >50-mM NaCl. The obtained results agreed with those reported in Hordeum vulgare [68], Salvinia molesta [69], Cornus sericea [70], and melons [19]. Salt stress induced the degradation of β-carotene, which further resulted in a decreased carotenoid content [71]. Carotenoids are integrated constituents of thylakoid membranes, and they absorb and transfer light to Chl and, thus, protect Chl from photooxidation [72]. Accordingly, the degradation of carotenoids might cause Chl degradation as well. The degradation of photosynthetic pigments under salinity is probably the consequence of increased toxic ions, enhanced chlorophyllase enzyme activity, and the damaging of the photosynthetic apparatus [20]. Recent research showed that a high level of toxic ions originating from different salts in wastewater induced the loss of photosynthetic pigments, including Chl a, Chl b, and carotenoids in wheat [73]. Considering that salinity caused an altered photosynthetic pigment metabolism, it is clear that the Chl content is strictly regulated under abiotic stress [7].
Chl a is present in the reaction centres of photosystem I and II, while Chl b is present exclusively in the pigment antenna system [74]. The weight of the Chl a/Chl b ratio is an important indicator of the photosynthetic apparatus adaptation to the light conditions [75]. The Chl a/Chl b ratio in dicotyledonous plants is usually 3:1 [76]. In the present study, there was a tendency toward an increased Chl a/Chl b ratio with the increased salinity level in all the analysed centaury shoots. In the non-transformed shoots, in the range of the NaCl concentrations, the Chl a/Chl b ratio ranged between 0.88 and 8.72 µg/g FW; in transgenic line AtCKX1-29, the detected ratio was 0.95-9.14; in AtCKX2-17, 2. 41-8.40; and in AtCKX2-26, 0.71-4.45. An increased Chl a/Chl b ratio was also observed in the control and transgenic AtCKX2 leaves of tobacco [49]. Additionally, salt stress increased the Chl a/Chl b ratio in pepper [77]. During the process of Chl degradation, Chl b is converted to Chl a, and this might explain the increment of the Chl a/Chl b ratio [78]. Traditionally, the sun-shaded tolerance in plants has been defined based on the Chl a/Chl b ratio [79,80]. The Chl a/Chl b ratio has variable values, and when the ratio is~3, it is clear that plants are adapting to the light conditions. The plants species living in the shadow showed Chl a/Chl b ratios lower than in the sun-exposed plants [40]. Thus, a decrease in the Chl a/Chl b ratio might be interpreted as an enlargement of photosystem II. Considering that Chl a is placed in photosystem I and II, it can be supposed that Chl a is probably primarily degraded during senescence. A stronger decrement of the Chl a content in comparison to Chl b and, accordingly, a reduced Chl a/Chl b ratio was also observed during leaf senescence in Hordeum vulgare leaves [81] and during dark-induced senescence in Cucurbita pepo cotyledons [82]. Previous investigations have also demonstrated darknessinduced cotyledon senescence in Arabidopsis seedlings [83]. Furthermore, it was indicated that CKs could serve as a regulatory mechanism coordinating senescence in individually darkened cotyledons [84]. Additionally, the in vitro culture conditions, although constantly controlled, enable light flux alterations in comparison to nature, and plants need to adapt to lowered light conditions. Thus, the ability of the whole plant to control the senescence progression underlies the coordination of senescence progression in individual leaves.
The weight ratio of the total Chl and total carotenoids is an indicator of the greenness of plants [40]. In a natural habitat, the Chl/carotenoids ratio is normally between 4.2 and 5 in leaves of sun-exposed plants and between 5.5 and 7.0 in leaves of shade-exposed plants. Lower values of the Chl/carotenoids ratio represent an indicator of the damage of the plant photosynthetic apparatus, which is expressed by a faster breakdown of Chl than carotenoids. Leaves with a Chl/carotenoids ratio between 2.5 and 3.5 are usually a yellow-green colour, and these leaves are experiencing senescence progress [85]. All the centaury plants used in this study were grown in vitro. The addition of NaCl in the medium reduced the Chl/carotenoids ratio in the centaury shoots. In the non-transformed shoots, in the range of the NaCl concentrations, the Chl/carotenoids ratio ranged between 14.84 and 1.61; in the transgenic line AtCKX1-29, the detected ratio was 18.05-1.22; in AtCKX2-17, 5.59-1.10; and in AtCKX2-26, 16.49-1.30. Based on the value of the Chl/carotenoids ratio in the non-transformed and AtCKX centaury plants grown in the NaCl-free medium, it can be assumed that the transgenic plants had a delayed senescence. Previous investigations have shown that the transgenic AtCKX leaves of A. thaliana and tobacco remained green even longer than the leaves of the non-transformed plants and, consequently, did not show an accelerated but, contrary to expectations, showed a delayed senescence [28,49]. Obviously, the decrement of CKs is certainly one of the preconditions, but not the initial signal, for the beginning of senescence. On the other hand, a decreased value of the Chl/carotenoids ratio in the non-transformed, as well as in the transgenic AtCKX, centaury shoots grown in a range of NaCl concentrations could be an indicator of accelerating the senescence process in the salt stress conditions. The increased values of the Chl a/Chl b ratio and, at the same time, decreased value of the weight Chl/carotenoids ratio are caused by the high irradiance adaptation response of the photosynthetic pigment apparatus of sun leaves [86].

Conclusions
Non-transformed and transgenic AtCKX centaury lines have different photosynthetic pigment contents as a response to salt stress conditions. Contrary to the theoretical expectations, the transgenic AtCKX centaury leaves did not show accelerated senescence, although the photosynthetic pigment contents decreased with the increasing salinity in the culture media. According to our results, it is clear that a low content of CKs in transgenic AtCKX centaury lines has a very important role in the progression of salt-induced senescence, but it is not a major signal definitively triggering its onset. However, the effect of salinity on photosynthetic pigments is highly specific and certainly requires additional investigation in order to provide an improved understanding of the pigment content variations among transgenic centaury plants.
Supplementary Materials: The following are available online et https://www.mdpi.com/article/ 10.3390/agronomy11102056/s1, Table S1: Photosythetic pigments ratios in the shoots of nontransformed and AtCKX transgenic centaury lines after 8 weeks of growth in 1 /2MS medium supplemented with graded NaCl concentration. The data represent the mean ± standard error. Means marked with a different letter significantly different from the control, according to the LSD test (p ≤ 0.05).

Conflicts of Interest:
The authors declare no conflict of interest.