Phenotype of L. davidii var. unicolor plant. (A) Phenotypes of L. davidii var. unicolor plants with different levels of yellowing. (B) Normal plants in the field. (C) Yellow plants in the field.
Variation in the photosynthetic pigments in the leaves of L. davidii var. unicolor. (A) Chlorophyll a. (B) Chlorophyll b. (C) Carotenoids. (D) Total chlorophyll. Different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).
Contents of chlorophyll synthesis precursors in leaves of L. davidii var. unicolor of different yellowing grades. (A) 5-Aminolevulinic acid. (B) Porphobilinogen. (C) Urogen III. (D) Coprogen III. (E) Protoporphyrin (Proto) IX. (F) Magnesium protoporphyrin (Mg-Proto) IX. (G) Pchlide A. (H) Chlorophyll a. (I) Chlorophyll b.
Influence of yellowing on the photosynthetic parameters of L. davidii var. unicolor leaves. (A) Photosynthetic rate. (B) Transpiration rate. (C) Stomatal conductance. (D) Saturated vapor pressure. (E) Intercellular CO2 concentration.
Influence of leaf yellowing on chlorophyll fluorescence parameters in leaves of L. davidii var. unicolor of different yellowing grades. (A) Maximal fluorescence (Fm). (B) Maximal photochemical efficiency of PS II (Fv/Fm). (C) Actual photosynthetic quantum yield [Y(II)]. (D) Photochemical quenching (qP). (E) Nonphotochemical quenching (NPQ). (F) Photosynthetic electron transport rate (ETR).
Correlation analysis of photosynthesis parameters and chlorophyll fluorescence.
Leaf anatomical structure of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2= seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y1; Y2 = anthesis of Y2.
Parameters of the leaf microstructure. (A) Leaf thickness. (B) Main vein thickness. (C) Upper epidermis thickness. (D) Lower epidermis thickness. (E) Palisade tissue thickness. (F) Spongy tissue thickness. (G) The ratio of palisade thickness to leaf thickness (CTR). (H) The ratio of palisade tissue thickness to spongy tissue thickness (PST).
Ultrastructure of mesophyll cells and chloroplasts of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2 = seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y01; Y2 = anthesis of Y2. Chl = chloroplast; CM = chloroplast membrane; CW = cell wall; GL = stroma thylakoid; GR = grana thylakoid; IS = intercellular space; M = mitochondria; N = nucleus; OD = osmiophilic droplet; Pb = plastoglobulus; RER = rough endoplasmic reticulum; SG = starch grain; V = vacuole.
Parameters of the leaf ultrastructure. (A) Number of chloroplasts. (B) Length of chloroplasts. (C) Width of chloroplasts. (D) Number of starch grains. (E) Length of starch grains. (F) Width of starch grains. (G) Number of plastoglobuli. (H) Diameter of plastoglobuli.
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Lilium davidii var. unicolor cotton is a famous edible lily with large-scale cultivation in China. To determine the cause of leaf yellowing in L. davidii var. unicolor, the photosynthetic characteristics and leaf structure of plants at different yellowing levels were studied. The results revealed that the chlorophyll content in the leaves of L. davidii var. unicolor decreased significantly as the degree of yellowing increased. Variation in the content of chlorophyll precursors revealed that in yellow-leafed plants, chlorophyll synthesis was impeded at the stage when coprogen is converted into Proto IX. Compared with those of normal plants, the thicknesses of the leaves, upper epidermis, palisade tissue, and spongy tissue of yellow-leafed plants were significantly lower. Distinct plasmolysis was observed in mesophyll cells. The cytomembrane and tonoplast were damaged. The number of chloroplasts and starch grains in the mesophyll cells of yellow-leafed plants decreased. The volume of chloroplasts also decreased, and structural damage occurred. Granum lamella failed to stack into granum, which led to a decrease in or disappearance of granum thylakoid. The variation in chloroplast structure and the reduction in chlorophyll content led to a further decrease in photosynthesis in yellow-leafed L. davidii var. unicolor plants. The photosynthetic parameters [net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr)] and chlorophyll fluorescence parameters [maximum quantum efficiency of photosystem II (PSII) (Fv/Fm), photochemical quantum yield of PSII [Y(II)], and electron transport rate (ETR)] decreased significantly. The Fv/Fm, Y(II), ETR, and photochemical chemical quenching were significantly positively correlated with Pn, Gs, and Tr, but highly negatively correlated with intercellular CO2 concentration (Ci) and vapor pressure saturation deficit (VPD). The results of this study provide important information for further studies of the response mechanism of plants to leaf yellowing. In addition, this study provides a theoretical basis for the prevention and recovery of yellow-leafed plants, which are important for increasing the yield and quality of L. davidii var. unicolor bulbs.
Lilium davidii var. unicolor cotton, which belongs to the genus Lilium, is one of the most important edible lilies. Bulbs of L. davidii var. unicolor have both edible and medicinal value. Therefore, it is used as a special vegetable in China and can be eaten fresh or cooked (Tang et al. 2021). Its bulbs can help strengthen the immune system because they are rich in nutrients such as starch, dietary fiber, vitamins, and various bioactive substances (You et al. 2010; Zhang et al. 2010). L. davidii var. unicolor has a long cultivation history in China and is propagated mainly by bulb division. With the extension of cultivation time and expansion of production areas, the yellowing phenomenon of the plant has increased substantially. The types of yellowing observed include dispersive yellowing and large-scale yellowing. According to the preliminary survey of our research group, at present, dispersive yellowing is common in the major production areas of L. davidii var. unicolor, whereas large-scale yellowing is becoming increasingly severe. Leaf yellowing of L. davidii var. unicolor directly causes plant growth restriction, early death, and a reduction in bulb yield and quality, thus dampening the morale of farmers and affecting the development of the L. davidii var. unicolor industry.
Plant yellowing during the growing season can be divided into two types: pathological yellowing and physiological yellowing (Liu et al. 2018). Pathological yellowing is caused mainly by pathogen infections (Dovas et al. 2002; Maust et al. 2003). Pathogens such as viruses infect roots, leaves, stems, bulbs, and other plant organs, disrupt their normal metabolic processes, and destroy organ structure. This leads to the appearance of leaf yellowing or chlorosis accompanied by cespitose stems and mottled leaves (Fan et al. 2019; Lee et al. 2007; Niimi et al. 2003; Zhang et al. 2015). Physiological yellowing is relatively common in lily production. Unfavorable environmental factors such as a lack or imbalance of nutrient elements, extreme temperatures, drought, flooding, and inappropriate lighting can cause physiological yellowing (Vacek et al. 2009). For example, the successive cultivation of lilies in the same field will cause continuous cropping obstacles, which will lead to growth restriction and plant yellowing. L. davidii var. unicolor plants that presented yellowing symptoms in the growing season also died earlier than normal plants did. Many studies related to plant yellowing have focused on investigating the causes (Lecoq et al. 1992; Navas-Castillo et al. 2000; Wu et al. 2007) and recovery approaches (Fan and Mattheis 2000). However, few studies have investigated the variations in the physiological and biochemical characteristics of yellow L. davidii var. unicolor plants.
Leaf color depends on the content, proportion, and distribution of pigments in the leaves (Reinbothe and Reinbothe 1996). Chlorophyll is the dominant pigment in the leaves of most green plants. A decrease in chlorophyll content causes variation in leaf color. Physiological studies of Chrysanthemum morifolium have shown that the yellow color of leaves is caused by chlorophyll loss (Shao et al. 2022). Wang et al. (2017) reported that the leaf chlorophyll content in yellow-leafed Forsythia cultivars is significantly lower than that in green-leafed cultivars. Zhang et al. (2019) reported that the total chlorophyll and carotenoid contents in yellow leaves of yellow tea plants were significantly lower than those in yellow and green leaves of normal plants. The chlorophyll content not only is an important indicator of yellow plants but also reflects the degree of photosynthesis of the plant. The content of chlorophyll depends on the dynamic balance between its synthesis and degradation (Rüdiger 1997; Yu et al. 2006). Although the biosynthesis pathways of chlorophyll have been clearly studied, its degradation pathway remains incompletely understood (Büchert et al. 2011; Eckardt 2009; Schelbert et al. 2009). The process of chlorophyll biosynthesis in higher plants is a complicated process in which 5-aminolevulinic acid (ALA), porphobilinogen, uroporphyrinogen III (urogen III), coprogen II, protoporphyrin IX (Proto IX), magnesium-protoporphyrin (Mg-Proto), and protochlorophyllide (Pchlide) are important precursors (Eckhardt et al. 2004; Tripathy and Pattanayak 2012). Generally, the first step is the formation of ALA. The ALA molecules are condensed to urogen III, which is oxidatively converted to Proto IX and then forms Mg-Proto and Pchlide (Beale 1999). By analyzing the variation in the contents of chlorophyll and its biosynthetic precursors, it can be preliminarily inferred that the chlorophyll biosynthesis process is normal. Drought stress causes a metabolic imbalance between chlorophyll and its precursors in Avena sativa, in which the content of ALA increases while the contents of porphobilinogen, Proto IX, Mg-Proto, and Pchlide decrease (Liu et al. 2022).
Measurements of leaf photosynthesis parameters and chlorophyll fluorescence provide an approach for exploring the photosynthetic capacity and efficiency of plants (Angert 2006). Variations in chlorophyll fluorescence parameters such as minimal fluorescence (Fo), maximal fluorescence (Fm), maximum quantum efficiency of photosystem II (PSII) (Fv/Fm), Fv/Fo, effective photochemical quantum yield of PSII [Y(II)], photochemical chemical quenching (qP), and non-photochemical quenching (qN) are indicators of a plant’s photosynthetic capacity. The chlorophyll fluorescence parameters Fv/Fm, Y(II), qP, and qN activities of a yellow leaf mutant of tomato were lower than those of its wild type under different temperature treatments (Ji et al. 2024). Similar variation was also found in yellow ‘Cabernet Sauvignon’ grapes (Huang et al. 2020). The measurement of parameters such as Fv/Fm is a relatively easy approach used to determine the degree of variation in photosynthesis before a plant presents visible symptoms (Willits and Peet 1999). Generally, the variations in chlorophyll fluorescence parameters signify that plants protect their photosynthetic reaction center by adjusting energy dissipation.
The leaf structure and chlorophyll content of yellow-leafed plants are significantly different from those of green-leafed plants (Xu et al. 2023). Generally, the chlorophyll content of yellow-leafed plants is lower than that of green-leafed plants (Wang et al. 2017). A decrease in chlorophyll content usually accompanies abnormalities in chloroplast structure. Zhang et al. (2023) reported defects in the leaf structure of a Brassica napus yellow leaf mutant in which mesophyll cells were out of shape and irregularly arranged. The palisade tissue and spongy tissue were loosely arranged. Similar results were reported by Xiao et al. (2013) for Brassica napus L. Chang et al. (2019) reported that the leaf microstructure of a tree peony (Paeonia suffruticosa) yellow-leaf mutant was similar to that of green leaves. However, there was a significant difference in the chloroplast ultrastructure between the green-leafed and yellow-leafed mutants. The stacks of grana disappeared from the chloroplasts of the yellow-leafed mutant, and only a few stromal thylakoid membranes remained along with clusters of osmiophilic granules. The structure of the thylakoid membranes in these chloroplasts was extremely disordered. Li et al. (2024) reported that the chlorophyll content in the leaves of an eggplant (Solanum melongena L.) yellow-leafed mutant was significantly lower than that in green leaves. The membrane structure of the chloroplasts was damaged, and the chloroplasts of the yellow-leafed mutant nearly disintegrated. The grana lamella was uneven and irregularly stacked. Many studies have shown that etiolation can seriously affect the photosynthesis of plants and further cause yield reduction (Chen et al. 2014; Liu et al. 2018; Luo et al. 2022). Although plant yellowing occurs in a large area of L. davidii var. unicolor production, few studies have been performed to reveal the variations in the physiological and biochemical characteristics of yellow plants. In this study, L. davidii var. unicolor plants were divided into three different yellowing groups (Y0, Y1, and Y2). Leaf photosynthetic pigments, photosynthesis characteristics, chlorophyll fluorescence parameters, and chlorophyll precursors were determined for plants with different yellowing levels. Moreover, the microstructures of the leaves as well as the ultrastructures of mesophyll cells and chloroplasts have revealed the relationship between leaf structure and yellowing. The aims of this study were to determine the variation in photosynthesis in yellowing plants of L. davidii var. unicolor, elucidate the relationship between leaf structure and yellowing, and understand the cause of leaf yellowing in L. davidii var. unicolor at the physiological, biochemical, and cytological levels, which could provide a theoretical basis for further studies of the mechanism of the response to yellowing. In addition, the results of this study could provide a reference for the prevention and recovery of yellow plants, which are important for increasing the yield and quality of L. davidii var. unicolor bulbs.
Bulbs of L. davidii var. unicolor were planted in Bingling Mountain village of Sanhe town, Ping’an District of Haidong City, Qinghai Province, China (lat. 36°25.8′N, long. 101°59.4′E, altitude 2591.6 m), in Mar 2020. The previous crop of the field was maize. After 1 year of growth, the large-scale yellowing of the plants occurred. In May 2021, L. davidii var. unicolor plants, which were cultivated for 2 years, were divided into three yellowing groups (Y0, Y1, and Y2) based on the visual observations and the chlorophyll content of the leaves (Table 1, Fig. 1). The plants were transplanted with soil (one bulb per pot) in polyethylene pots with signage in the terrace of the Agricultural and Animal Husbandry Experimental Building of Qinghai University (lat. 36°43.32′N, long. 101°45.2′E, altitude 2310 m) with 30-cm spacing for each pot (23 cm diameter, 13 cm height). The plants were watered thoroughly and covered with a sunshade net for 1 week after transplanting, and routine management was performed once per week. The plants were grown in the same environment and unified cultivation management was applied. The annual average temperature in 2021 was 8.23 °C. The annual precipitation was 476.76 mm. The parameters of leaf photosynthetic characteristics during different growth periods were determined from Apr to Sep 2022.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The chlorophyll content was determined with 80% acetone according to the methods of Arnon (1949). The absorbance (osmiophilic droplet) value was obtained via ultraviolet spectrophotometry (UV-2012C; Unico Instrument Co., Ltd., China) at wavelengths of 665 nm (A665), 649 nm (A649), and 470 nm (A470). The contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll (chlorophyll a+b) were calculated.
In Apr 2022, young leaves were collected to measure the content of chlorophyll synthesis precursors. Three individual plants were sampled from each level of yellowing. Three replicates were performed for each plant. The ALA was extracted and quantified according to the methods of Dei (1985). Porphobilinogen, uroporphyrinogen III (urogen III), and coproporphyrinogen III (coprogen III) levels were determined according to the method of Bogorad (1962). The Proto IX, Mg-Proto IX, and Pchlide a levels were determined according to the approach of Liu et al. (2015).
A portable photosynthesis measurement system GFS-3000 (Heinz Walz GmbH, Effeltrich, Germany) was used to determine photosynthetic parameters from 9:00 AM to 12:00 AM on a sunny day in Aug 2022. The light intensity was set at 1500 μmol·m−2·s−1, the flow rate was 750 m·s−1, the leaf chamber area was 4 cm2, and the impeller width was 7. The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and water vapor pressure saturation deficit (VPD) were determined. All measurements were performed on the leaves of the middle part of each plant. Three individual plants were measured for each yellowing level. Three replicates were performed for each plant.
Chlorophyll fluorescence was assessed in vivo via pulse-amplitude modulation (Junior-PAM; Walz, Effeltrich, Germany) according to the methods of Motohashi and Myouga (2015). The plants were dark-adapted for 30 min before the assessment of minimal fluorescence (Fo). The maximal fluorescence (Fm) was measured with a saturation pulse at 10,000 µmol·m−2·s−1 for 0.6 s. The maximum quantum efficiency of PSII (Fv/Fm) was determined as follows: Fv/Fm= (Fm-Fo)/Fm. The Y(II), photosynthetic electron transport rate (ETR), qP, and nonphotochemical quenching (NPQ) were obtained under actinic light (125 µmol·m−2·s−1). Three plants were randomly selected from each yellowing level. At least three leaves on the middle part of each plant were selected to determine the chlorophyll fluorescence parameters.
Three plants were randomly selected from each grade of yellowing. The sixth to eighth leaves from the top of each plant were selected for this experiment. To observe the microstructure of the leaf, the middle part of each leaf (5 mm × 5 mm) was sampled. The plant tissue samples were refined, dehydrated, paraffin-embedded, microsectioned, red–solid green-stained, and subjected to other treatments by Nanjing Shuaipu Biological Company. Three different vision fields were randomly selected, and each vision field was repeated three times to measure the leaf thickness, thickness of the leaf main vein, thickness of the upper epidermis, thickness of the lower epidermis, thickness of the palisade tissue, and thickness of the spongy tissue. The ratio of palisade thickness to leaf thickness (CTR) was calculated (Li et al. 2018). The ratio of palisade tissue thickness to spongy tissue thickness (PST) was also calculated (Liu et al. 2018). To observe the ultrastructure of the chloroplast, the middle part of each leaf (1 mm × 1 mm) was sampled, and an ultrathin section was made. The dehydrated samples were embedded in epoxy resin and cut into 60- to 80-nm-thick sections. The ultrathin sections were stained with 2% uranium acetate saturated alcohol solution for 15 min, followed by lead citrate for 15 min. Finally, the ultrathin sections were examined under an HT7700 (HITACHI, Tokyo, Japan) electron microscope, and images were taken.
An analysis of variance was performed using IBM SPSS Statistics 22, and significant differences between treatments were determined using the least significant difference test at P < 0.05. The results of the Pearson correlation analysis and the correlation heatmap were plotted using Origin software (version 9.8.5.204).
The results revealed that in the leaves of normal plants (Y0), the content of photosynthetic pigments was lowest at the seedling stage (Fig. 2). It increased with plant growth and reached its highest value at the anthesis stage. The content of photosynthetic pigments decreased gradually as the plants withered. In the Y1 plants, the variation in the photosynthetic pigment content was the same as that in the Y0 plants. The rate of increase in the content of photosynthetic pigments was lower than that in the Y0 plants. Therefore, the contents of chlorophyll and carotenoids in Y1 plants were significantly lower than those in Y0 plants from anthesis to the withering stage. From the visible flower bud to the withering stage, the photosynthetic pigment content in the leaves of Y2 plants was extremely significantly lower than that in the leaves of Y0 and Y1 plants. No significant variation was observed among the different growth stages.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The contents of the chlorophyll precursors ALA, porphobilinogen, urogen III, and coprogen III in yellow plants were greater than those in normal plants (Fig. 3). Proto IX is the oxidative product of coprogen III. Proto IX in yellow-type plants was extremely significantly lower than that in normal plants, which indicated that the metabolic process converting coprogen III to Proto IX was blocked. The contents of Mg-Proto IX and Pchlide in Y1 and Y2 plants were significantly lower than those in Y0 plants.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The Pn is a key indicator of photosynthesis. As shown in Fig. 4, during the plant growth and development periods, the variation in the Pn of plants of different yellowing grades was similar. From the seedling stage to the anthesis stage, the Pn increased significantly and reached its highest value. As the plants withered, the photosynthetic rates declined rapidly. A comparison of the Pn of different yellow-leafed plants revealed that the Pn of yellow-leafed plants (Y1 and Y2) was extremely significantly lower than that of normal plants (Y0). Furthermore, the Pn of Y2 was extremely significantly lower than that of Y1.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
Stomatal conductance represents the degree of stomata opening. It influences plant transpiration, respiration, and photosynthesis. In normal L. davidii var. unicolor plants, the Gs and Tr were lowest at the seedling stage. Both Gs and Tr increased rapidly as the plant grew and reached their highest values at the anthesis stage. However, the rates of increase in Gs and Tr in yellow plants were lower than those in normal plants. At the seedling stage, the Gs and Tr of Y0 were significantly greater than those of Y1 and Y2. Moreover, the Gs and Tr of Y1 were significantly greater than those of Y2. At the seedling and anthesis stages, the Gs and Tr of Y1 and Y2 were significantly lower than those of Y0. However, the Gs and Tr of Y1 and Y2 continued to increase until the withering stage. Therefore, no significant variation was detected among plants with different degrees of yellowing at the withering stage.
The variation in the water VPD was consistent with the variation in the Pn in the leaves of L. davidii var. unicolor. Leaf yellowing induced a significant increase in VPD. From the seedling stage to the anthesis stage, the VPD of the yellow plants was significantly greater than that of the normal plants. At the withering stage, the VPD of Y2 was significantly greater than the VPD of the Y1 and Y0 plants.
The Ci is the ratio of the CO2 assimilation rate to stomatal conductance. The Ci of normal plants remained relatively stable, whereas the Ci of yellow plants fluctuated significantly. In both the Y1 and Y2 plants, the Ci decreased significantly from the seedling stage to the anthesis stage, but then it increased. The Ci of the Y1 and Y2 plants was significantly greater than that of the Y0 plants at the seedling stage. However, at the seeding and anthesis stages, the Ci of the Y1 and Y2 plants was lower than that of the Y0 plants. At the withering stage, the Ci of Y2 was significantly greater than the Ci of Y0 and Y1.
The results revealed that leaf yellowing significantly influenced the chlorophyll fluorescence parameters (Fig. 5). The Fv/Fm and Y(II) are the maximum and effective photosynthetic efficiencies of PSII, which reflect the potential and effective efficiency of light energy conversion in plants, respectively. During the growth of L. davidii var. unicolor, the Fv/Fm and Y(II) ratio increased from the seedling stage to the anthesis stage. The Fv/Fm ratio of yellow-leafed plants dramatically decreased to low levels. Significant variation was detected in the plants from the different yellowing treatments. The Y(II) of yellow-leafed plants (Y1 and Y2) was significantly lower than that of normal plants (Y0) at the seeding and anthesis stages. The Fv/Fm and Y(II) of the Y2 plants were the lowest at each stage, indicating that the PSII reaction center was severely damaged (Hu et al. 2023). The ETR of normal plants (Y0) was significantly greater than that of Y1 and Y2 plants (P < 0.05) at the seedling stage. These findings indicated that plant yellowing significantly reduced the electron transfer rate and potential photosynthetic capacity in the leaves of L. davidii var. unicolor at the beginning of plant growth.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The decrease in fluorescence reflecting photosynthetic activities is called qP. Like that of the ETR, the qP of the yellow plants was significantly lower than that of the normal plants at the seedling stage. This result indicates that the photochemical conversion efficiency of the leaves decreased when the plants began yellowing. The thermal dissipation of the absorbed light energy of PSII is called NPQ. The results revealed that there was no significant variation in NPQ in plants of different yellowing grades at the seedling stage. The NPQ of Y1 plants increased significantly beginning at the seedling stage and was significantly greater than that of Y0 and Y2 plants at the visible flower bud and anthesis stages. These findings indicate that the heat dissipation efficiency of the Y1 plants was greater than that of the Y0 and Y2 plants.
The Pn reflects the ability of plants to accumulate organic matter through photosynthesis. It is influenced by the chlorophyll content and chlorophyll fluorescence. A correlation analysis revealed that the Pn was significantly correlated with the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids (Fig. 6). The photosynthetic parameters were significantly correlated with each other. Strong positive correlations were found between Pn, Gs, Tr, and Ci. Strong negative correlations were found between the VPD and other photosynthetic parameters. Among the chlorophyll fluorescence parameters, strong positive correlations were found between Fm, Fv/Fm, and Y(II). The ETR was significantly correlated with qP and NPQ. A correlation analysis of the photosynthetic and chlorophyll fluorescence parameters revealed that Fm, Fv/Fm, and Y(II) were strongly positively correlated with the photosynthetic parameters Pn, Tr, Gs, and Ci, but highly negatively correlated with VPD.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The palisade cells of yellow leaves became shorter and smaller, and the spongy cells were no longer full (Fig. 7). The cavity between the spongy cells decreased. The boundary between palisade tissue and spongy tissue is not clear. The leaf microstructure-related parameters revealed that the thickness of the leaf and the palisade tissue and spongy tissue of yellow-leafed plants were significantly lower than those of normal plants at the seedling stage. Compared with that of the control, the leaf thickness of Y1 and Y2 decreased by 17.9% and 19.6%, respectively, at the anthesis stage. Moreover, the palisade tissue thicknesses of Y1 and Y2 decreased by 35.8% and 43.9%, respectively, compared with that of the control. Compared with that of normal plants, the spongy tissue thickness of yellow-leafed plants decreased by 16.5%. The ratio of palisade thickness to leaf thickness and the ratio of palisade tissue thickness to spongy tissue thickness of yellow-leafed plants were significantly lower than those of normal plants beginning at the visible flower bud stage (Fig. 8).
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The appearance of mesophyll cells differed among yellow-leafed plants. The thickness of the cell wall was not uniform, and partial damage and atrophy appeared in the cytomembrane. The phenomenon of plasmolysis appears in the cells. Moreover, the number of organelles was lower, the vacuole membrane was damaged, and the number of mitochondrial cristae decreased or even disappeared (Fig. 9).
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
The number of chloroplasts in the mesophyll cells of yellow-leafed plants decreased, the volume of chloroplasts decreased, and structural damage occurred (Fig. 10). In Y2 plants, granum lamella failed to stack into granum, which led to a decrease in or disappearance of granum thylakoid. At the seedling and anthesis stages, the number of chloroplasts in Y1 and Y2 was lower than that in Y0. The width of chloroplasts in Y2 was significantly lower than that in Y0. The length of chloroplasts in Y1 and Y2 was significantly lower than that in Y0 at the anthesis stage. The number of starch grains in the mesophyll cells of yellow-leafed plants was significantly lower than that in the mesophyll cells of normal plants at all stages. The length and width of starch grains in the mesophyll cells of yellow-leafed plants were significantly smaller than those of starch grains in normal plants at the anthesis stage. The number of plastoglobuli in Y2 was significantly smaller than that in Y1 and Y0 at the seedling stage. However, it was significantly greater than Y1 and Y0 at the visible flower stage. The number of plastoglobuli increased significantly from the seedling stage to the visible flower stage, indicating that obvious accumulation of plastoglobuli occurred in the Y2 plants.
Citation: HortScience 60, 4; 10.21273/HORTSCI18377-24
Changes in plant leaf color are related to the synthesis and degradation of pigments in leaves and the structure and quantity of chloroplasts. The content of photosynthetic pigments is generally lower than that of the corresponding wild type when the leaf color of the plant is yellow, etiolated, or mutated. For example, the pigment content in the yellow area of Aucuba japonica is significantly lower than that in the green area (Zhang et al. 2018). In this study, the contents of the photosynthetic pigments chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the leaves of yellow L. davidii var. unicolor plants were significantly lower than those in normal plants during the whole growth and development process. A study of chlorophyll synthesis precursors revealed that the process of chlorophyll synthesis in yellow plants was impeded at the step of coprogen III conversion to Proto IX. Blocked synthesis of chlorophyll precursor substances affects the normal synthesis of chlorophyll in leaves, resulting in a lower chlorophyll content, which the leads to changes in leaf color. Currently, blocked sites for chlorophyll synthesis have been identified in a variety of plant leaf color mutants, and the same blocking site has been found in the xantha mutant of Oncidium (Wang et al. 2013), the chlorophyll-deficient mutant of Brassica juncea (Lv et al. 2010), the variegated leaves of Iris ensata (Zeng et al. 2024), and the chlorophyll-less mutant of Lagerstroemia indica (Wang et al. 2017). However, different chlorophyll synthesis blocking sites have been found in different leaf mutants of Cymbidium hybrids (Zhong et al. 2021).
Yellowing of external morphology occurs under the control of changes in leaf tissue structure and internal cells (Liu et al. 2014). In this study, we observed the leaf anatomical structure of yellow plants of L. davidii var. unicolor and reported that the leaf thickness, upper epidermal thickness, spongy tissue thickness and palisade tissue thickness of yellow plants were significantly lower than those of normal plants, which is in line with the findings of Sun et al. (2020) and Zhang et al. (2022) on Forsythia. Cheng et al. (2022) studied the chloroplast structure and photosynthesis of tomato yellow mutant leaves and reported that compared with those of green leaves, the chloroplast structure of tomato yellow mutant leaves was obviously disrupted, and the photosynthetic capacity was weakened. Xu (2023) reported that more chloroplasts were present in normal plant leaves than in yellow mutant leaves, and significant differences in the arrangement and number of thylakoids were also detected between normal plants and yellow leaf mutants. The thylakoids in normal plants were arranged neatly, concentrated, and numerous, whereas the thylakoids in the yellow leaf mutant were smaller, fewer, thinner, and disordered. In this study, we found that yellowing reduced the number of chloroplasts, which made them smaller, that the basal lamellar system could not stack to form basal granules, and that the lamellar structure of stroma-like vesicles was blurred, which led to a decrease in the photosynthetic capacity of the leaves. In addition, the length of starch grains was significantly reduced in yellow plants, whereas the number of plastid microspheres increased with the increasing degree of yellowing. Starch granules are produced by photosynthetic products that have not had time to be transported out of the chloroplasts, and the decrease in the number of starch granules indicates that the number of photosynthetic products decreases in the leaves of the plants. Plastid microspheres are generally a product of chloroplast-like vesicle membrane degradation, and an increase in plastid microspheres indicates damage to the membrane system of chloroplasts (Prakash et al. 2001; Smith et al. 2000). In terms of whole leaf pulp cells, the greater the degree of yellowing, the greater the degree of plasma wall separation of the leaf pulp cells, and the cell membrane and vesicle membrane of the leaf pulp cells of the yellow plants exhibited different degrees of breakage, indicating that the yellow plants were no longer able to perform normal osmotic regulation.
The photosynthetic capacity of plants is positively correlated with the content of chlorophyll in leaves. Generally, a decrease in chlorophyll content reduces plant photosynthesis (Sairam and Srivastava 2002). The results of this study revealed that the photosynthetic parameters Pn, Gs, and Tr in yellow-leafed L. davidii var. unicolor plants were significantly lower than those in normal plants. Similar results were reported for a rice chlorophyll-deficient mutant and Schefflera odorata cv. variegata (Tan et al. 2019; Wu et al. 2022). Strong positive correlations were found between Pn, Gs, Tr, and Ci, consistent with the results of studies of yellowed grapes and cherries (Huang et al. 2020; Jiang et al. 2022). As an intermediary for photosynthesis in plants, Ci not only is restricted by the CO2 concentration in the atmosphere but also is influenced by stomatal conductance and photosynthetic consumption in leaves. At the seedling stage of L. davidii var. unicolor, the Ci of yellow-leafed plants was greater than that of the control. At this time, the photosynthetic rate of yellow-leafed plants was lower than that of normal plants. This result indicates that the consumption of CO2 by yellow leaves was lower than that by green leaves, which led to higher Ci values. As plants grow, intercellular CO2 is used. Moreover, the stomatal conductance of yellow leaves was lower than that of green leaves, which means that less CO2 was absorbed. Therefore, after the seedling stage, the Ci of yellow-leafed plants began to decrease and was lower than that of the control. Generally, a decrease in the photosynthetic rate of plants is caused by “stomatal limitation” or “nonstomatal limitation” (Farquhar and Sharkey 1982; Lawlor 2002). In this study, the GS and chlorophyll contents of yellow-leafed plants were lower than those of normal plants, and the decrease in leaf GS was accompanied by increases in Ci and VPD, which indicate that the decreased photosynthetic rate of L. davidii var. unicolor was caused by “nonstomatal limitation.” Therefore, the low chlorophyll content was the main reason for the decrease in the photosynthetic rate of L. davidii var. unicolor.
Chlorophyll fluorescence parameters are important indicators that can reflect the photosynthetic activities of plants (Rascher et al. 2000). The Fv/Fm reflects the light energy conversion efficiency of the PSII reaction center. The results of this study revealed that the Fv/Fm and Y(II) of yellow-leafed plants decreased significantly, which was consistent with the findings of a study of Oryza sativa L. (Wang et al. 2015). However, the photosynthetic efficiency of the yellow-green leaf mutant (ygl) of rice was improved because the light absorbed by the mutant was more efficiently partitioned to photosynthesis (Wang et al. 2022). The Fv/Fm is considered an important parameter for evaluating the level of photosynthetic stress and photoinhibition (Malapascua et al. 2014). Strong positive correlations were found between Fm, Fv/Fm, and Y(II). This finding is consistent with the research results of winter wheat (Jia et al. 2019). Generally, the Fv/Fm of most plants is between 0.80 and 0.85. However, it decreases significantly when stress or injury occurs (Roy et al. 2024). In this study, the Fv/Fm values of the Y1 and Y2 plants were 0.72 ± 0.01 and 0.62 ± 0.01, respectively, which were lower than normal. These findings indicated that yellow-leafed L. davidii var. unicolor plants were in a state of photoinhibition and that their light energy conversion ability decreased. This finding is consistent with the results reported for Schefflera odorata cv. Variegataso (Wu et al. 2022), Sinobambusa tootsik f. luteoloalbostriata (Chen et al. 2019), Lycopersicon esculentum (Yang et al. 2018), and grapevines (Shahsavandi et al. 2020). In L. davidii var. unicolor, the ETR of yellow-leafed plants was significantly lower than that of the control before flowering. These findings indicated that in the early stage of L. davidii var. unicolor development, the reduction in the chlorophyll content in leaves reduced the electron transfer rate. Chlorophyll harvests solar energy and drives electrons to transfer light energy to the photosynthetic reaction center (Tanaka and Tanaka 2006). It is believed that a high chlorophyll content could intercept and absorb more light energy (Ort et al. 2015). However, a low chlorophyll content does not always reduce the photosynthetic rate. A study of a pale green-leafed rice mutant suggested that a reduction in chlorophyll content resulted in a significant increase in photosystem II efficiency and ETR (Gu et al. 2017).
Heat dissipation is an important protection strategy when plants are under photoinhibition. Photochemical quenching represents the level of photosynthetic activity of plants, whereas nonphotochemical quenching reflects the ability of plants to dissipate excess light energy into heat, which is the ability of light protection. This study revealed that the NPQ of yellow-leafed plants was greater than that of normal plants. These findings indicate that yellow-leafed plants cannot use excess light energy when the external environment is the same. Therefore, yellow-leafed plants presented better light protection ability, which they achieve by increasing heat dissipation.
The chlorophyll content in the leaves of L. davidii var. unicolor decreased significantly as the degree of yellowing increased. During the process of chlorophyll synthesis, the conversion step from coprogen to Proto IX is hindered, leading to a decrease in chlorophyll. By observing the microstructure of the leaves and the ultrastructures of mesophyll cells and chloroplasts, abnormal structures were found in yellow-leafed plants. It was deduced that the yellowing of L. davidii var. unicolor occurred because the chlorophyll synthesis stage was blocked and the chloroplast structure was abnormal. The variation in chloroplast structure and the reduction in chlorophyll content further led to a decrease in the photosynthetic capacity of yellow-leafed L. davidii var. unicolor plants. The photosynthetic parameters (Pn, Gs, and Tr) and chlorophyll fluorescence parameters (Fv/Fm, Y(II), and ETR) decreased significantly. Fv/Fm, Y(II), ETR, and qP were significantly positively correlated with Pn, Gs, and Tr, but highly negatively correlated with Ci and VPD, which indicated that the PSII reaction center of yellow leaves was damaged, and that the photochemical conversion efficiency of the leaves decreased.
Phenotype of L. davidii var. unicolor plant. (A) Phenotypes of L. davidii var. unicolor plants with different levels of yellowing. (B) Normal plants in the field. (C) Yellow plants in the field.
Variation in the photosynthetic pigments in the leaves of L. davidii var. unicolor. (A) Chlorophyll a. (B) Chlorophyll b. (C) Carotenoids. (D) Total chlorophyll. Different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).
Contents of chlorophyll synthesis precursors in leaves of L. davidii var. unicolor of different yellowing grades. (A) 5-Aminolevulinic acid. (B) Porphobilinogen. (C) Urogen III. (D) Coprogen III. (E) Protoporphyrin (Proto) IX. (F) Magnesium protoporphyrin (Mg-Proto) IX. (G) Pchlide A. (H) Chlorophyll a. (I) Chlorophyll b.
Influence of yellowing on the photosynthetic parameters of L. davidii var. unicolor leaves. (A) Photosynthetic rate. (B) Transpiration rate. (C) Stomatal conductance. (D) Saturated vapor pressure. (E) Intercellular CO2 concentration.
Influence of leaf yellowing on chlorophyll fluorescence parameters in leaves of L. davidii var. unicolor of different yellowing grades. (A) Maximal fluorescence (Fm). (B) Maximal photochemical efficiency of PS II (Fv/Fm). (C) Actual photosynthetic quantum yield [Y(II)]. (D) Photochemical quenching (qP). (E) Nonphotochemical quenching (NPQ). (F) Photosynthetic electron transport rate (ETR).
Correlation analysis of photosynthesis parameters and chlorophyll fluorescence.
Leaf anatomical structure of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2= seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y1; Y2 = anthesis of Y2.
Parameters of the leaf microstructure. (A) Leaf thickness. (B) Main vein thickness. (C) Upper epidermis thickness. (D) Lower epidermis thickness. (E) Palisade tissue thickness. (F) Spongy tissue thickness. (G) The ratio of palisade thickness to leaf thickness (CTR). (H) The ratio of palisade tissue thickness to spongy tissue thickness (PST).
Ultrastructure of mesophyll cells and chloroplasts of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2 = seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y01; Y2 = anthesis of Y2. Chl = chloroplast; CM = chloroplast membrane; CW = cell wall; GL = stroma thylakoid; GR = grana thylakoid; IS = intercellular space; M = mitochondria; N = nucleus; OD = osmiophilic droplet; Pb = plastoglobulus; RER = rough endoplasmic reticulum; SG = starch grain; V = vacuole.
Parameters of the leaf ultrastructure. (A) Number of chloroplasts. (B) Length of chloroplasts. (C) Width of chloroplasts. (D) Number of starch grains. (E) Length of starch grains. (F) Width of starch grains. (G) Number of plastoglobuli. (H) Diameter of plastoglobuli.
Contributor Notes
This work was supported by the Qinghai Province Science and Technology Department (2023-NK-151) and Kunlun Talent Science and Technology Leading Talent plan of Qinghai Province Program.
N.T. is the corresponding author. E-mail: natasha_tn@163.com.
Phenotype of L. davidii var. unicolor plant. (A) Phenotypes of L. davidii var. unicolor plants with different levels of yellowing. (B) Normal plants in the field. (C) Yellow plants in the field.
Variation in the photosynthetic pigments in the leaves of L. davidii var. unicolor. (A) Chlorophyll a. (B) Chlorophyll b. (C) Carotenoids. (D) Total chlorophyll. Different uppercase letters indicate highly significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).
Contents of chlorophyll synthesis precursors in leaves of L. davidii var. unicolor of different yellowing grades. (A) 5-Aminolevulinic acid. (B) Porphobilinogen. (C) Urogen III. (D) Coprogen III. (E) Protoporphyrin (Proto) IX. (F) Magnesium protoporphyrin (Mg-Proto) IX. (G) Pchlide A. (H) Chlorophyll a. (I) Chlorophyll b.
Influence of yellowing on the photosynthetic parameters of L. davidii var. unicolor leaves. (A) Photosynthetic rate. (B) Transpiration rate. (C) Stomatal conductance. (D) Saturated vapor pressure. (E) Intercellular CO2 concentration.
Influence of leaf yellowing on chlorophyll fluorescence parameters in leaves of L. davidii var. unicolor of different yellowing grades. (A) Maximal fluorescence (Fm). (B) Maximal photochemical efficiency of PS II (Fv/Fm). (C) Actual photosynthetic quantum yield [Y(II)]. (D) Photochemical quenching (qP). (E) Nonphotochemical quenching (NPQ). (F) Photosynthetic electron transport rate (ETR).
Correlation analysis of photosynthesis parameters and chlorophyll fluorescence.
Leaf anatomical structure of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2= seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y1; Y2 = anthesis of Y2.
Parameters of the leaf microstructure. (A) Leaf thickness. (B) Main vein thickness. (C) Upper epidermis thickness. (D) Lower epidermis thickness. (E) Palisade tissue thickness. (F) Spongy tissue thickness. (G) The ratio of palisade thickness to leaf thickness (CTR). (H) The ratio of palisade tissue thickness to spongy tissue thickness (PST).
Ultrastructure of mesophyll cells and chloroplasts of yellow-leafed L. davidii var. unicolor plants. (A) Y0 = seedling stage of Y0; Y1 = seedling stage of Y1; Y2 = seedling stage of Y2. (B) Y0 = visible flower bud of Y0; Y1 = visible flower bud of Y1; Y2 = visible flower bud of Y2. (C) Y0 = anthesis of Y0; Y1 = anthesis of Y01; Y2 = anthesis of Y2. Chl = chloroplast; CM = chloroplast membrane; CW = cell wall; GL = stroma thylakoid; GR = grana thylakoid; IS = intercellular space; M = mitochondria; N = nucleus; OD = osmiophilic droplet; Pb = plastoglobulus; RER = rough endoplasmic reticulum; SG = starch grain; V = vacuole.
Parameters of the leaf ultrastructure. (A) Number of chloroplasts. (B) Length of chloroplasts. (C) Width of chloroplasts. (D) Number of starch grains. (E) Length of starch grains. (F) Width of starch grains. (G) Number of plastoglobuli. (H) Diameter of plastoglobuli.
