Effects of Dark Treatment on Lignin and Cellulose Synthesis in Celery

: To clarify the impact of continuous dark stress on lignin and cellulose synthesis in celery, shade-tolerant celery varieties were screened. Yellow celery variety ‘Qianhuang No.1’ and green celery variety ‘Qianlv No.1’ were separately grown in vegetable greenhouses. Dark treatments were applied using PVC shading sleeves for 4, 8, 12, and 16 d after celery had grown 10–13 true leaf blades. This study aimed to investigate the impact of varying periods of dark treatment on the morphological characteristics, lignin accumulation, and cellulose accumulation in celery. The results showed that dark treatment led to celery yellowing, a reduced stem thickness, and an increased plant height. Analysis of lignin and cellulose contents, as well as the expression of related genes, showed that dark treatment caused down-regulation of AgLAC , AgC3 ′ H , AgCCR , AgPOD and AgCAD genes, leading to changes in lignin accumulation. Dark treatment inhibited the expression of the AgCesA6 gene, thus affecting cellulose synthesis. Under dark conditions, the expression of AgF5H and AgHCT genes had little effect on lignin content in celery, and the expression of the AgCslD3 gene had little effect on cellulose content. Analysis of morphological characteristics, lignin accumulation and cellulose accumulation after different lengths of dark treatment demonstrated that ‘Qianlv No.1’ is a shade-tolerant variety in contrast to ‘Qianhuang No.1’.


Introduction
Celery (Apium graveolens L.) is a perennial herbaceous plant of the Apiaceae family.It is native to the Mediterranean region and is now widely cultivated in Europe, East Asia, Sweden and other swampy regions [1].Celery is a highly nutritious vegetable, rich in fiber, minerals and bioactive compounds such as lignin, cellulose, potassium, calcium and magnesium, as well as apigenin, phenols, coumarins and volatile oils [2].It has numerous health benefits, including lowering blood pressure [3], reducing inflammation [4], improving digestive function and promoting cardiovascular health [5].
Lignin and cellulose are important components of dietary fiber and are naturally present in fruits and vegetables [6][7][8].During plant growth and development, lignin deposition in the cell walls of the xylem increases their thickness and enhances the hardness and toughness of stems, providing internal mechanical support for plants to withstand Agronomy 2024, 14, 896 2 of 15 abiotic and biotic stress [9,10].Lignification is the most direct indicator of plant senescence and significantly affects the taste and quality of vegetables.Studies have shown a strong negative correlation between lignin content and edible quality, such as lignification causing peas to become hard, rough and fibrous, affecting their taste [11].Cellulose, as an important component of insoluble dietary fiber (IDF), affects the quality and taste of vegetables depending on its content [12].
Blanching culture is a special cultivation technique to make some vegetables grow under dark or weak light conditions and form soft and yellowing organs.It is a facility cultivation method to promote chlorophyll degradation.Blanching culture affects the accumulation of nutrients and active substance biosynthesis in vegetables, thus changing the appearance of crops and improving the flavor of crops.The types of softening planting mainly include chive (Allium tuberosum Rottler ex Spreng.),water dropwort (Oenanthe javanica (Blume) DC.), garlic (Allium sativum L.), shallot (Allium cepa L.) and celery (A.graveolens L.) [20][21][22][23][24].However, the molecular effect of blanching culture under dark conditions on lignin synthesis in celery has not been fully studied.Guizhou Province of China has a subtropical humid monsoon climate with more precipitation, an obvious rainy season, more cloudy days and less sunshine.Therefore, studying the effect of softening culture on lignin biosynthesis in celery can not only guide the application of celery etiolation cultivation, but also screen shade-tolerant celery varieties suitable for planting in Guizhou.
In this research, to investigate the metabolism of lignin and cellulose in celery during dark treatment, 12 genes involved in lignin metabolism pathways identified from the celery transcriptome database, as well as 2 genes, AgCslD3 and AgCesA6, involved in cellulose metabolism pathways, were analyzed [25].The lignin distribution in the petiole was qualitatively assessed using histochemical methods.The lignin and cellulose contents and the expression profiles of related genes were analyzed during the dark treatment period.The results of this study will contribute to elucidating the changes in lignin and cellulose during celery cultivation and identifying shade-tolerant celery varieties.

Celery Material
The celery varieties 'Qianlv No.1' and 'Qianhuang No.1' were used to study the effects of dark treatment on lignin and cellulose.'Qianlv No.1' and 'Qianhuang No.1' were planted in a plastic vegetable greenhouse at the Institute of Horticulture, Guizhou Academy of Agricultural Sciences (106.67 • E, 26.51 • N).The seeds were sown in plastic basins and then the seedlings were transplanted into pots containing stroma of organic soil and vermiculite (2:1; v/v) in December 2022.The experiment used celery plants with 10~13 true leaf blades as the test material.The celery seedlings with 10~13 true leaf blades were covered with an opaque, black PVC sleeve for dark treatment, and conventional cultivation in natural light served as the control treatment.The plants were subjected to the dark treatment and control treatment for 0, 4, 8, 12, and 16 d, respectively.The petioles and leaf blades of dark and control treatments in 'Qianlv No.1' and 'Qianhuang No.1' were frozen with liquid nitrogen and stored at −80 • C for subsequent experiments.Meanwhile, petioles from the dark and control treatments were fixed in a 70% FAA (formalin-glacial acetic acid with 70% ethanol; 1:1:18; v/v) fixative solution and stored at 4 • C for paraffin sectioning and fluorescence micrographs.Three biological replicates were performed for each experimental treatment material.

Determination of Lignin and Cellulose Content
The lignin and cellulose contents of celery samples were determined by using lignin and cellulose content assay kits and ultraviolet spectrophotometry (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).Lignin and cellulose were extracted and detected by the method described in the kit's instructions.The phenolic hydroxyl groups in lignin have a characteristic absorption peak at 280 nm after acetylation, and the light absorption value at 280 nm is positively correlated with the lignin content.The content of cellulose was determined using an anthrone chromogenic agent under strong acidic conditions.
Therefore, wavelengths of 280 nm and 620 nm were, respectively, used for the lignin and cellulose content measurements via T-UV1810S spectrophotometry (Shanghai Yoke Instruments Meters Co., Ltd., Shanghai, China).Glacial acetic acid and distilled water were, respectively, used as blank controls to detect the absorbance to determine the content of lignin and cellulose.

Preparation and Histochemical Staining of Paraffin Sections
The transverse sections of the stem were used for safranin o-fast green staining and microscopy experiments.The petioles from the dark and control treatments were fixed with FAA fixative solution for 24 h and dehydrated with different ethanol gradients.The petioles were processed for paraffin embedding and sectioning.The samples were cut into 4 µm × 4 µm slices using a paraffin microtome.Paraffin slides were successively put into two portions of environmentally friendly dewaxing transparent liquid for 20 min, two portions of pure ethanol for 5 min, and 75% ethanol for 5 min, and the slides were then kept in tap water.The sections were stained with safranin O staining solution for 2 h, then rinsed with tap water to remove excess dye.The slices were placed into 50%, 70%, 80% gradient alcohol for decolorization for 3~8 s each.The slices were stained with the plant solid green dye solution for 6~20 s and dehydrated with anhydrous ethanol in three times.Finally, the sections placed put into three cylinders of xylene for 5 min before they were observed under a microscope; images were taken and then analyzed.The cytoderm which had lignified appeared red and the color of cellulose cell wall was green.

Total RNA Isolation and Real-Time Fluorescence Quantitative PCR Analysis
The relative expression of related genes was detected via qRT-PCR.Total RNA was extracted from petioles and leaf blades with Trizol reagent and treated with DNase I (Vazyme Biotech Co., Ltd., Nanjing, China).RNA quality was detected using agarose gel electrophoresis.RNA quality and purity were assessed using the OD260/280 ratio which was determined via a Nanodrop 2000 spectrophotometer (Implen GmbH, München, German).cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China).
Table 1.Primers for RT-qPCR related to lignin and cellulose synthesis.

Statistical Analysis
SPSS software (IBM SPSS Statistics for Windows, version 26.0) was used to conduct one-way ANOVAs at the 0.05 level for all data with significant differences.

Morphological Characteristics of Celery during Dark Treatment and Conventional Cultivation
The petioles and leaf blades of celery, compared with those from conventional cultivation, had different characteristics after different dark treatment (Figure 1).Before dark treatment, celery grew well; the leaf blades and petioles of 'Qianhuang No.1' were light yellow, and the leaf blades and petioles of 'Qianlv No.1' were dark green (Figure 1 commercial properties of the plants were completely lost; the old leaf blades 'Qianhuang No.1' were obviously yellow, the new petioles had turned white, and t plants were weak (Figure 1(E1,E2)).The new petioles of 'Qianlv No.1' plants were wh the leaf blades were curled and drooping, and the plants were obviously overgrown a weak (Figure 1(E3,E4)).

Lignin and Cellulose Contents of Celery during Dark Treatments and Control Conditions
The effects of different stages of dark treatments on lignin contents were compar During the control period, the lignin content in the leaf blades of 'Qianhuang No showed a significant increase followed by a decrease, reaching the highest level after 8 The lignin content in the leaf blades of 'Qianlv No.1' did not decrease significantly.Duri the dark treatment period, the lignin content in the leaf blades of 'Qianhuang No slightly increased at first and then gradually decreased, while the lignin content in the l blades of 'Qianlv No.1' did not decrease significantly.During the treatment, the lign content in the petioles of 'Qianhuang No.1' in the experimental group and the cont group showed a trend of increasing significantly first and then decreasing sharply.T lignin content in the petioles of 'Qianlv No.1' in the control group increased significan at first, then decreased and increased again, while the lignin content in the dark treatme group decreased significantly after 4 d and then remained relatively stable.Overall, t lignin content in the dark treatment group was lower than that in the control group, a the lignin content in the leaf blades during the dark treatment stage did not decrea significantly but was maintained at a relatively constant level (Figure 2).

Lignin and Cellulose Contents of Celery during Dark Treatments and Control Conditions
The effects of different stages of dark treatments on lignin contents were compared.During the control period, the lignin content in the leaf blades of 'Qianhuang No.1' showed a significant increase followed by a decrease, reaching the highest level after 8 d.The lignin content in the leaf blades of 'Qianlv No.1' did not decrease significantly.During the dark treatment period, the lignin content in the leaf blades of 'Qianhuang No.1' slightly increased at first and then gradually decreased, while the lignin content in the leaf blades of 'Qianlv No.1' did not decrease significantly.During the treatment, the lignin content in the petioles of 'Qianhuang No.1' in the experimental group and the control group showed a trend of increasing significantly first and then decreasing sharply.The lignin content in the petioles of 'Qianlv No.1' in the control group increased significantly at first, then decreased and increased again, while the lignin content in the dark treatment group decreased significantly after 4 d and then remained relatively stable.Overall, the lignin content in the dark treatment group was lower than that in the control group, and the lignin content in the leaf blades during the dark treatment stage did not decrease significantly but was maintained at a relatively constant level (Figure 2).
first and then increased significantly.The cellulose content in the petioles of 'Qianlv No.1' showed a non-significant increase first and then decrease, while the cellulose content in the dark treatment group increased significantly after 8 d and then decreased significantly.Overall, after 4 d of treatment, the cellulose content in the dark treatment group was lower than that in the control group, and the dark treatment of 'Qianhuang No.1' showed a significantly lower cellulose content than the control group (Figure 3).The cellulose content of control and dark treatment plants at different periods were compared.During the control period, the cellulose content in the leaf blades of 'Qianhuang No.1' gradually increased significantly, while the cellulose content in the leaf blades of 'Qianlv No.1' first increased significantly and then slowly decreased.During the dark treatment period, the cellulose content in the leaf blades of 'Qianhuang No.1' decreased steadily after a stable period and then increased significantly, while the cellulose content in the leaf blades of 'Qianlv No.1' increased first and then decreased.Under dark treatment, the cellulose content in the petiole of 'Qianhuang No.1' decreased first and then increased significantly.The cellulose content in the petioles of 'Qianlv No.1' showed a non-significant increase first and then decrease, while the cellulose content in the dark treatment group increased significantly after 8 d and then decreased significantly.Overall, after 4 d of treatment, the cellulose content in the dark treatment group was lower than that in the control group, and the dark treatment of 'Qianhuang No.1' showed a significantly lower cellulose content than the control group (Figure 3).The cellulose content of control and dark treatment plants at different periods were compared.During the control period, the cellulose content in the leaf blades of 'Qianhuang No.1' gradually increased significantly, while the cellulose content in the leaf blades of 'Qianlv No.1' first increased significantly and then slowly decreased.During the dark treatment period, the cellulose content in the leaf blades of 'Qianhuang No.1' decreased steadily after a stable period and then increased significantly, while the cellulose content in the leaf blades of 'Qianlv No.1' increased first and then decreased.Under dark treatment, the cellulose content in the petiole of 'Qianhuang No.1' decreased first and then increased significantly.The cellulose content in the petioles of 'Qianlv No.1' showed a non-significant increase first and then decrease, while the cellulose content in the dark treatment group increased significantly after 8 d and then decreased significantly.Overall, after 4 d of treatment, the cellulose content in the dark treatment group was lower than that in the control group, and the dark treatment of 'Qianhuang No.1' showed a significantly lower cellulose content than the control group (Figure 3).

Histochemical Analysis of Lignin Distribution in Celery Petioles
The present study utilized safranin staining solution to identify the distribution of lignin in celery petioles.The results showed that lignin was mainly distributed in the xylem, and the color of cell walls changed over the treatment time in both the treatment and control groups (Figure 4).The color of the cell walls in the treatment group changed from purple-aubergine to light red to no red, while the color in the control group remained purple-aubergine and gradually became lighter red.In addition, the number of xylem cells increased and their arrangement became tighter during the treatment process.After 8 d of treatment, the depth of red color in cell walls was different between the treatment and control groups (Figure 4(A-III-D-III)), and after 16 d, the cell wall of the celery petioles in the control group was light red, while there was no red color in the cell walls of the treatment group (Figure 4(A-V-D-V)).These results suggest that the lignin in celery petioles was mainly concentrated in xylem, and the treatment time has a certain impact on the distribution of lignin.

Histochemical Analysis of Lignin Distribution in Celery Petioles
The present study utilized safranin staining solution to identify the distribu lignin in celery petioles.The results showed that lignin was mainly distributed xylem, and the color of cell walls changed over the treatment time in both the trea and control groups (Figure 4).The color of the cell walls in the treatment group ch from purple-aubergine to light red to no red, while the color in the control group rem purple-aubergine and gradually became lighter red.In addition, the number of cells increased and their arrangement became tighter during the treatment process 8 d of treatment, the depth of red color in cell walls was different between the trea and control groups (Figure 4(A-III-D-III)), and after 16 d, the cell wall of the celery p in the control group was light red, while there was no red color in the cell walls treatment group (Figure 4(A-V-D-V)).These results suggest that the lignin in petioles was mainly concentrated in xylem, and the treatment time has a certain i on the distribution of lignin.

Expression Profiles of Lignin and Cellulose Metabolism-Related Genes in Celery durin Dark Treatment and Control Conditions
To further validate the relationship between the expression of genes invol lignin and cellulose synthesis after dark treatment, we identified the lignin synthesis AgF5H, AgC4H, AgHCT, Ag4CL, AgCCR, AgPAL, AgPOD, AgCAD, AgCCoAOMT, Ag

Expression Profiles of Lignin and Cellulose Metabolism-Related Genes in Celery during Dark Treatment and Control Conditions
To further validate the relationship between the expression of genes involved in lignin and cellulose synthesis after dark treatment, we identified the lignin synthesis genes AgF5H, AgC4H, AgHCT, Ag4CL, AgCCR, AgPAL, AgPOD, AgCAD, AgCCoAOMT, AgC3 ′ H, AgCOMT and AgLAC, as well as the cellulose synthesis genes AgCesA6 and AgCslD3 from the celery database.
For the leaf blades of celery variety 'Qianhuang No.1', the expression of the lignin synthesis genes AgPAL, AgC4H, Ag4CL, AgCCoAOMT, AgCAD and AgF5H showed an initial increase followed by a decrease, while the expression of AgHCT and AgCCR gradually increased.The expression of AgPOD and AgCOMT decreased gradually.The expression of the AgLAC gene showed no significant change under dark treatment, but increased gradually under control conditions.The expression of the AgC3 ′ H gene increased gradually under dark treatment, while it initially increased and then decreased under control conditions.Compared to the control, the expression levels of AgC3 ′ H and AgCOMT genes were significantly lower under dark treatment, while AgF5H and AgHCT expression was significantly higher than the control.The expression of the cellulose synthesis genes AgCesA6 and AgCslD3 increased gradually, with AgCslD3 showing a more pronounced trend.The expression levels of AgC4H, Ag4CL, AgPAL, AgCAD, AgCCoAOMT and AgCOMT genes were highest after 12 d of treatment (Figure 5).For the leaf blades of celery variety 'Qianlv No.1', the expression of the lignin synthesis genes AgPAL, AgC4H, Ag4CL, AgHCT, AgCCR and AgF5H showed an initial increase followed by a decrease, while the expression of AgCAD, AgCCoAOMT and AgCCR gradually increased, and AgPOD expression gradually decreased.The expression of the AgC3 ′ H gene showed no significant change under dark treatment, while it initially increased and then decreased under control conditions.The expression of the AgLAC gene showed no significant change before 12 d of dark treatment, but significantly decreased after 16 d, while it increased dramatically under control conditions.The expression of the cellulose synthesis gene AgCesA6 decreased gradually, while AgCslD3 initially increased and then decreased.The expression levels of AgF5H, AgC4H, AgHCT, Ag4CL, AgCCR and AgPAL genes were highest at 8 d of treatment (Figure 6).For the stems of celery variety 'Qianhuang No.1', the expression of the lignin synthesis genes AgPAL, AgF5H, AgHCT and AgC3'H gradually increased, while AgC4H, Ag4CL, AgCCR, AgCAD and AgCCoAOMT showed a slight decrease followed by an increase, and AgLAC and AgCOMT decreased gradually.The expression of the AgPOD gene changed the most after 12 d of dark treatment, with no significant changes in other stages.The expression of the cellulose synthesis gene AgCslD3 showed a trend of first decreasing and then increasing, while AgCesA6 gradually decreased and showed higher expression compared to the control under dark treatment.The expression levels of the genes AgF5H, AgC4H, AgHCT, Ag4CL, AgCCR, AgPAL, AgCAD, AgCCoAOMT, AgCOMT and AgCslD3 were highest at 12 d of dark treatment, with AgF5H and AgHCT showing significantly higher expression levels than the control (Figure 7).
For the stems of celery variety 'Qianlv No.1', the expression of genes AgF5H, AgPAL, AgC4H, Ag4CL, AgHCT, AgCAD, AgCCoAOMT, AgC3 ′ H and AgCOMT showed an initial increase followed by a decrease, while AgPOD and AgLAC showed an increasing trend, and AgCCR showed a decreasing trend.The expression of the cellulose synthesis gene AgCslD3 decreased dramatically, while AgCesA6 showed an initial increase followed by a decrease.The expression levels of the genes AgC4H, AgHCT, Ag4CL, AgPAL, AgCAD, AgCCoAOMT, AgCOMT and AgC3 ′ H were highest after 8 d of dark treatment (Figure 8).According to the expression analysis results of lignin-synthesis-related genes and cellulose synthesis genes in the leaf blades of 'Qianhuang No.1' and 'Qianlv No.1' and the petioles of 'Qianhuang No.1' and 'Qianlv No.1', we can summarize the expression trend of lignin-synthesis-related genes as follows: the genes AgPAL, AgC4H, Ag4CL, AgCAD, AgCCoAOMT, and AgF5H first increase and then decrease; the expression of AgHCT and AgCCR gradually increases; the expression of AgPOD and AgCOMT gradually decreases; the expression of AgLAC shows no obvious change under dark treatment; and the expression of AgC3 ′ H gradually increases.The expression trend of cellulose synthesis genes is as follows: the expression of AgCesA6 and AgCslD3 gradually increases, with AgCslD3 showing a more dramatic increase.Overall, the lignin and cellulose contents during the dark treatment stage were lower than those in the control group, and the lignin content in the leaf blades during the dark treatment stage remained relatively stable, while the cellulose content fluctuated within a certain period of time.The dark treatment had a more significant effect on the leaf blades of 'Qianhuang No.1' than on 'Qianlv No.1'.

Discussion
Huang et al.'s study showed that light plays a broad role in regulating a plant's growth, morphology, and physiological metabolism [26].When plants exhibit a shade avoidance response, their stems elongate and their diameter decreases.Studies have shown that shading increases internode elongation in rice, which increases the risk of lodging [27,28].In this study, the color of celery leaf blades and petioles gradually became lighter and turned yellow to white as the duration of the dark treatment increased.After 8 d of dark treatment, the commercial quality of celery gradually deteriorated, with new leaf blades curling and lightening in color and new petioles significantly reducing in diameter and elongating.This is consistent with previous studies that showed a decrease in the red light and far-red light (R/FR) ratio, inhibiting soybean stem lateral growth, promoting internode elongation at the base of the stem, and resulting in a reduced stem thickness and an increased plant height [29,30].Previous studies have shown that light can regulate gibberellin (GA) biosynthesis and metabolism, and GA plays an important role in leaf yellowing and stem elongation.Rice and wheat mutants with GA synthesis genes exhibit a dwarf phenotype [31][32][33].Light and GA have an antagonistic effect on cell elongation, where light can appropriately inhibit growth, while GA promotes yellowing and growth [34].Based on this, we speculate that the color change in celery is primarily influenced by photosynthesis.Dark treatment impedes photosynthesis, preventing the normal synthesis of pigments.Celery can appropriately inhibit plant growth under normal light, while dark treatment may induce changes in GA synthesis, resulting in yellowing, a decreased stem thickness, and an increased plant height.This further confirms the impact of light and light quality on plant morphogenesis.
Dietary fiber is an important factor that affects the quality, texture and flavor of vegetables, and celery leaf blades and petioles are rich in dietary fiber.Lignin and cellulose are important components of dietary fiber and play a crucial role in plant support and disease resistance.Research has shown that light intensity and quality can affect the activity of lignin synthesis, thereby significantly influencing the biosynthesis and metabolism of lignin [34], e.g., lignin accumulation in soybeans is significantly inhibited under shady conditions [35].Darkness also has a certain effect on cellulose deposition [36].
In this study, compared to the control group, the lignin content in celery leaf blades and petioles decreased in the dark treatment.Moreover, the 'Qianhuang No.1' variety showed a significant reduction in the lignin content in leaves after 4, 8 and 16 d of treatment, as well as a significant reduction in petiole lignin content after 12 and 16 d.However, the 'Qianlv No.1' variety only showed a significant reduction in petiole lignin content after 4 d of treatment.The changes in petiole lignin content were consistent with its distribution in the xylem, which is in line with previous findings that lignin content decreases in rice [37], tobacco [38], and tea plants [39] under low light conditions.This indicates that dark treatment leads to changes in celery's lignin content, and the 'Qianhuang No.1' variety is more sensitive (A1-A4)).After 4 d of dark treatment, the apparent change of plants was not obvious; the yellow color of old and new leaf blades of 'Qianhuang No.1' slightly deepened, and the petiole growth of 'Qianlv No.1' accelerated (Figure 1(B1-B4)).After 8 d of dark treatment, the yellow color of 'Qianhuang No.1' plants had obviously deepened, and the change in new leaf blades was the most obvious (Figure 1(C1,C2)).The 'Qianlv No.1' plant grew faster and began to turn yellow (Figure 1(C3,C4)).After 12 d of dark treatment, the merchantability of the plants gradually deteriorated; the new petioles of the 'Qianhuang No.1' plant had extended, the new leaf blades curled into deep yellow, and the plants were obviously overgrown (Figure 1(D1,D2)).The new leaf blades of the 'Qianlv No.1' plant clearly turned yellow (Figure 1(D3,D4)).After 16 d of dark treatment, the commercial properties of the plants were completely lost; the old leaf blades of 'Qianhuang No.1' were obviously yellow, the new petioles had turned white, and the plants were weak (Figure 1(E1,E2)).The new petioles of 'Qianlv No.1' plants were white, the leaf blades were curled and drooping, and the plants were obviously overgrown and weak (Figure 1(E3,E4)).

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14, x FOR PEER REVIEW 9 of 16during the dark treatment stage were lower than those in the control group, and the lignin content in the leaf blades during the dark treatment stage remained relatively stable, while the cellulose content fluctuated within a certain period of time.The dark treatment had a more significant effect on the leaf blades of 'Qianhuang No.1' than on 'Qianlv No.1'.