Physiological Mechanisms of BvCPD Regulation in Sugar Beet Growth

: Sugar beet is an important sugar crop, and its roots are mainly used for processing raw materials to produce products such as sugar, molasses, and saccharin, as well as being used as fodder for livestock. BvCPD , a key enzyme gene for brassinosteroid (BR) synthesis, regulates the development of parenchyma cells and vascular bundles by promoting BR synthesis, which promotes the expansion of the sugar beet taproot and influences the growth, development, and yield of sugar beets. This study investigated the impact of BvCPD on the physiological metabolism of sugar beet utilizing BvCPD overexpression, silent, and wild-type (WT) lines. BvCPD increased the chlorophyll content and maximum photochemical efficiency and improved the photosynthetic characteristics of sugar beet leaves. Simultaneously, BvCPD increased the rate of sugar beet taproot respiration and ATP content by enhancing the activities of phosphoglycerate kinase, alcohol dehydrogenase, sucrose synthase, and sucrose synthase catabolism. Moreover, BvCPD induced changes in the sugar fraction content, which increased the sugar yield of a single plant. In addition, BvCPD promoted water absorption, nitrogen accumulation, and lignin/cellulose synthesis activities, facilitated by increased activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, cellulose synthase, and protein serine/threonine phosphatases, providing the requisite energy and materials for sugar beet growth. These findings not only provide a new perspective for understanding the physiological mechanisms regulating the growth of sugar beets but also provide a theoretical basis for the future improvement of sugar beet varieties through molecular breeding techniques.


Introduction
Sugar beet is a diploid (2n = 18) biennial cash crop of the genus Beta L. in the Chenopodiaceae family.It is widely used in the food industry as the second most important sugar crop after sugarcane, and its roots are rich in sucrose [1].Therefore, taproot development is a major topic of interest in sugar beet research.As the sugar beet hypocotyl gradually differentiates to form the taproot, the activities of mineral elements and nucleic acid metabolism in the plant are enhanced.With the continuous differentiation of the root meristem and increase in the cambium, the cambium splits inward to form triarch xylem cells and splits outward to form triarch phloem cells, and it finally forms a triarch vascular bundle [2].The expansion of the taproot is partly due to the enlargement and amplification of parenchyma cells and partly due to the thickening of vascular rings [3].In recent years, many researchers have attempted to regulate the growth of sugar beet taproots by improving cultivation techniques, spraying nutrients, growth regulators, and phytohormones to enhance the yield and quality of sugar beets.For example, compared to furrow irrigation, infiltration irrigation increases the efficiency of water and nitrogen utilization in sugar beets, achieving both yield and quality improvements [4].Spraying 100 mg•L −1 zinc and 40 mg•L −1 molybdenum on the leaves of sugar beet can balance nutrient uptake and translocation, promoting sugar beet growth and yield [5].The application of organic products, such as humic acid, can modulate different biochemical effects, such as photosynthetic rate, cell membrane permeability, and cell elongation in sugar beets, which improves water utilization efficiency and promotes sugar beet yield [6,7].When melatonin is applied to sugar beets, in combination with foliar and root applications, it promotes taproot yields and sugar content by improving photosynthesis, water status, ionic homeostasis, and antioxidant defense systems [8].Foliar spraying of jasmonic acid significantly improves the mean values of all traits in sugar beets and increases the sugar content and yield of the taproot [9].Therefore, improvements in various cultivation practices can enhance the quality and yield of sugar beets through modulation of their physiological and metabolic levels.
Exogenous spraying with brassinolide significantly promotes sugar beet taproot expansion [3].Transcriptomic analyses have identified several genes associated with taproot expansion during sugar beet growth, including BvDof, BvBZR, BvXTH, and BvCPD.Notably, the BvCPD gene, encoding a key enzyme in brassinosteroid (BR) biosynthesis, is significantly upregulated during the taproot and sugar growth periods [10].BvCPD promotes the development of parenchyma cells and vascular bundles in the taproot of sugar beets by increasing the BR content and taproot diameter [11].Heterologous expression of the Populus PeCPD gene in Arabidopsis mutants results in elongated hypocotyls and roots, increased plant height, individual enlargement, broader leaves, enhanced xylem formation, greater pith cell numbers, augmented vascular bundle counts, and thickened stems [12].In addition, the overexpression of OsCPD1 and OsCPD2 in rice results in a typical BR-enhanced phenotype that plays an important role in maintaining plant structure by regulating BR biosynthesis and increasing leaf angle and seed size [13].These results indicate that CPD genes regulate plant growth and development.However, few studies have focused on the effects of CPD genes on plant physiological functions.
Our research group found that BvCPD promotes the growth of sugar beets, with the overexpression and silencing lines of BvCPD differing significantly from those of the wild-type (WT) phenotype.Overexpression of BvCPD leads to increased leaf length, leaf width, and leaf fresh weight, as well as enhanced petiole fresh weight in sugar beets.Moreover, it results in lateral thickening of the taproot and fibrous roots, contributing to a well-developed root system.Conversely, silencing the expression of this gene in sugar beets resulted in restricted leaf and petiole development, thin taproots, and sparse fibrous roots [11].However, the physiological metabolism regulated by BvCPD to promote sugar beet growth remains unknown.Therefore, we used sugar beet BvCPD overexpression and RNAi silencing lines to elucidate the functional effects of the gene on sugar beet leaf and taproot development.We analyzed changes in physiological indices, such as photosynthesis and respiration metabolism, sugar metabolism, water and nutrient uptake, and activities of cell growth-related enzymes, aiming to clarify the role of BvCPD in regulating sugar beet physiological metabolism.Our findings lay a foundation for studying the regulatory mechanism of the BvCPD gene in sugar beet taproots.

Plant Materials
The agrobacterium-mediated method was used to obtain BvCPD transgenic sugar beet.Studies were conducted using the sugar beet BvCPD overexpressing strain OE1-3, the RNAi silencing strain R1-3, and WT sugar beet.The expression of BvCPD in overexpression lines OE1-3 plants was higher than that of WT, but due to the different sites of gene insertion during the genetic transformation of the plants, the expression of BvCPD was elevated to a different extent in each overexpression line; conversely, the expression of BvCPD in silencing lines R1-3 plants was lower than that of WT, but the expression of BvCPD was reduced to a different extent in each silencing line.The acquisition and characterization of BvCPD transgenic lines have been published in previous studies [11].The plant materials were cultured in an artificial climate chamber set to 25 °C, 70% humidity, and an 18/6 h light/dark photoperiod.

Determination of Photosynthetic Performance
Determination of chlorophyll content: One month post-transplantation of transgenic plants, the fourth leaves from the flag leaf of each line were selected, with three biological replicates conducted.From these leaves, 1 g was carefully weighed away from the main leaf vein, and thin filaments were excised.The resulting mixture was immersed in a solution of acetone and anhydrous ethanol (1:1 ratio) to prevent light exposure.Subsequently, the leaves underwent agitation in a shaker at 25 °C and 110 rotations per min for 24 h.Absorption values were measured using a spectrophotometer (Shanghai Tianmei Scientific Instrument Co., Shanghai, China) at 645 nm and 633 nm to calculate the contents of total chlorophyll, chlorophyll A, and chlorophyll B, respectively.Chlorophyll A (mg.g −1 ) = (12.71× A663 − 2.59 × A645) × V/W; chlorophyll B (mg.g −1 ) = (22.88× A645 − 4.67 × A663) × V/W; and total chlorophyll (mg.g −1 ) = (8.04× A663 + 20.29 × A645) × V/W.Determination of net photosynthetic rate: An LI-6400 photosynthesizer (LI-COR, Lincoln, NE, USA) was utilized to assess the net photosynthetic (Pn) values.Specifically, the fourth leaves from the flag leaf of each line were employed for this analysis, with three biological replicates conducted.
Determination of maximum photochemical efficiency: A PAM-2500 portable pulse-modulated chlorophyll fluorometer (WALZ, Nuremberg, BAV, Germany) was employed to measure the Fv/Fm values, indicating the maximum photochemical efficiency.For this assessment, the fourth leaves from the flag leaf of each line were utilized, ensuring the exclusion of leaf veins.Measurements were taken after a 20 min dark treatment using specialized leaf clamps, with three biological replicates performed.

Determination of Respiration Rate
The respiration rate was determined using an infrared gas analyzer (YX-306B, Beijing Yuxiang Hengye Measurement and Control Technology Co., Ltd., Beijing, China).The taproot of each transgenic line was placed into containers, CO2 generation was measured, and minute-by-minute changes in CO2 production were documented, with three biological replicates conducted.The formula was calculated as follows: respiration rate = (C1 − C0)/W × t.C1 represents the amount of CO2 released at the time of measurement (mg), C0 represents the initial amount of CO2 released at the time of measurement (mg), W represents the fresh weight of beetroot tubers (g), and t represents the measurement time (h).

Determination of Moisture and Nitrogen Content
The water content and relative water content were determined in samples of sugar beet leaves, petioles, and taproots.The fresh weight (W1) of each sample was measured.Subsequently, the samples were completely immersed in distilled water for 24 h, after which the saturated fresh weight (W2) was recorded.Following immersion, the samples were dried at 85 °C for 12 h until a constant weight was achieved, and the dry weight (W3) was determined.The formulas for calculating water content (%) and relative water content (%) are as follows: water content (%) = (W1 − W3)/W3 × 100%, and relative water content (%) = (W1 − W3)/(W2 − W3) × 100%.Nitrogen content was determined according to the standard NY/T 2017-2011 for the determination of nitrogen, phosphorus, and potassium in plants [14].

Determination of ATP Content and Enzyme Activity
Eighty days post-sugar beet transplantation, samples for testing were harvested from cambium rings 1-4.These samples were carefully cut into fine filaments and then dried in an oven at 85 °C for 12 h to constant weight and ground into a powder for further analysis.The ATP content and enzymatic activities of various enzymes were assessed, including phosphoglycerate kinase (PGK), alcohol dehydrogenase (ADH), sucrose synthase (SS), sucrose synthase catabolism (SS-C), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), cellulose synthase (CesA), and protein serine/threonine phosphatases (PSP).These measurements were conducted using ELISA enzyme activity kits, according to the manufacturer's instructions (Jiangsu Su Enzyme Science and Technology Co., Ltd., Nanjing, China).

Determination of Sugar Fractions
The mother roots of each transgenic line were used to determine the sugar content, which was assessed using a Refractometer PAL-1 (Guangzhou Atago Scientific Instrument Co., Guangzhou, China).
(1) Extraction of soluble sugars: Mother roots from each transgenic line were utilized for analysis.BvCPD transgenic sugar beets and WT taproots were shredded and shade-dried, followed by oven drying at 105 °C for 30 min and then 85 °C for 12 h until a constant weight was attained.The dried samples were then ground into a powder.For extraction, 0.05 g of the powdered sample was weighed into a 10 mL centrifuge tube, 4 mL of 80% ethanol solution was added, and the mixture was placed in an 80 °C water bath for 40 min.After centrifugation, the supernatant was collected.The extraction process was repeated using 80% ethanol solution until a final volume of 10 mL was achieved, yielding the solution to be measured.(2) Determination of total soluble sugar content: Two milliliters of the solution to be measured was taken and mixed with 0.5 mL of anthrone ethyl acetate reagent (prepared by dissolving 1 g of anthrone in 50 mL of ethyl acetate solution) and 5 mL of concentrated sulfuric acid.The mixture was then subjected to a 100 °C water bath for 1 min, followed by cooling.The optical density (OD) value was determined at 625 nm.
(3) Determination of sucrose content: A volume of 150 μL of the solution to be measured was mixed with 150 μL of 2 mol/L NaOH and subjected to a 100 °C water bath for 5 min.After cooling, 2.1 mL of 10 mol/L HCl solution and 0.6 mL of 0.1% resorcinol were added, followed by thorough shaking.The mixture was then subjected to an 80 °C water bath for 10 min, and the OD value was determined at 480 nm.(4) Determination of fructose content: A volume of 0.4 mL of the solution to be measured was mixed with 2.8 mL of 2 mol/L HCl solution and 0.8 mL of 0.1% resorcinol.The mixture was subjected to an 80 °C water bath for 10 min, and the OD value was determined at 480 nm.(5) Determination of glucose content: The glucose content was determined according to the instructions of the glucose content detection kit (ADS-W-TDX002) manufactured by Jiangsu Edison Biological Co. in Nanjing, China.

Determination of Yield
The yield was measured according to the taproot cutting standard GB/T 10496-2018 [15].Cutting was performed 0.5 cm above the first leaf scar to determine the yield of the sugar beet taproot.

Statistical Analysis
Differences between means were assessed using SPSS 20 at the 5% and 1% probability levels.Statistical plots were generated using GraphPad Prism 6 software.

BvCPD Promotes Photosynthetic Properties of Sugar Beet Leaves
Photosynthesis has an important influence on the sugar yield of sugar beet taproots.To investigate the effects of BvCPD on the photosynthetic function of sugar beet leaves, chlorophyll A, chlorophyll B, total chlorophyll, net photosynthetic rate, and maximum photochemical efficiency were measured in transgenic sugar beet leaves.The results showed that chlorophyll A, chlorophyll B, and total chlorophyll levels were significantly higher in BvCPD overexpressing lines than in the WT lines (Table 1).Among the lines, the OE2 line exhibited the highest total chlorophyll content at 2.91 mg.g −1 fresh weight (FW), which represented a 31.91%increase over the WT, while the R2 line displayed the lowest total chlorophyll content at 1.61 mg.g −1 FW, which represented a 27.09% decrease compared to the WT.Similar trends were observed for chlorophyll A and B contents across the lines.However, the net photosynthetic rate (Pn) was not significantly different among the lines.Notably, the maximum photochemical efficiencies (Fv/Fm) of the BvCPD overexpressing lines were significantly increased by 2% compared to those of the WT, whereas those of the silenced lines were reduced by 1% to 4%.

BvCPD Promotes Energy Metabolism in Taproots
The respiration rates of the taproots from different BvCPD transgenic lines were measured.The results showed that BvCPD overexpression lines exhibited significantly higher rates compared to WT lines.The increase in respiration rates among the overexpression lines ranged from 16.67% to 21.49%, whereas the silenced lines showed a decrease ranging from 22.30% to 31.39% (Figure 1A).Moreover, ATP content determination within and between rings 1-4 of the vascular bundle cambium in sugar beet taproots revealed a similar trend.ATP content increased in cambium rings 1-4 of the overexpressing lines and decreased in the silenced lines (Figure 1B).Furthermore, we examined the activities of the PGK and ADH enzymes related to energy metabolism and showed that they were significantly elevated in each BvCPD overexpression line but reduced in the silenced line.These findings were consistent with the changing trend of ATP content in each line (Figure 1C,D).

BvCPD Regulates Sugar Metabolism in Sugar Beets
To clarify the regulatory effect of BvCPD on sugar metabolism in sugar beets, the sugar yield of sugar beet taproots was measured in a single plant after harvesting.Compared with the WT, the BvCPD overexpressing and silenced lines showed significantly or extremely significantly reduced sugar content in their taproots (Figure 2A).However, the single-plant taproot weights of the BvCPD overexpression lines were higher than those of the WT, and the taproot weights of the silenced lines were lower than those of the WT (Figure 2B).Except for BvCPD overexpression line OE3, all transgenic lines differed significantly from the WT.Therefore, although the BvCPD overexpression line showed a decreasing trend in single-plant sugar content, the single-plant sugar yield was not significantly reduced.Both OE1 and OE2 had significantly higher sugar yields than that of the WT, whereas the silenced line ultimately resulted in a highly significant reduction in single-plant sugar yields because of a significant decrease in both sugar yields and single-plant taproot weights (Figure 2C).These results indicate that although BvCPD overexpression may decrease the sugar content per plant, the expansion of taproots can compensate for this decrease, thereby ensuring that sugar production per plant is not significantly diminished but rather increased.To investigate the changes in sugar fractions in the BvCPD transgenic lines, the contents of total soluble sugar, sucrose, fructose, and glucose were determined in cambium rings 1-4 of the sugar beet taproots.The results showed that the total soluble sugar content in both BvCPD overexpressing and silenced lines was less than that in the WT, and the difference between the overexpressing lines and the WT became more significant as it extended toward the outer ring (Figure 2D).Sucrose, which accounted for the highest proportion of total soluble sugars, also showed a decreasing trend across all BvCPD overexpression lines, especially in cambium rings 2-4.Sucrose can be hydrolyzed to fructose and glucose.In all BvCPD overexpression lines, the fructose content was significantly increased while the glucose content was significantly decreased.In contrast, in the silenced lines, the total soluble sugar and sucrose contents decreased significantly, the fructose content did not change significantly, and only the glucose content increased significantly.These results suggest that overexpression or silent expression of BvCPD in sugar beets affects the content of each sugar component in cambium rings 1-4 of the taproot (Figure 2E-G).
We examined the activities of SS and SS-C in the vascular bundle cambium rings 1-4 of the taproots of each transgenic line and WT sugar beet.The results showed that the activities of SS and SS-C were significantly increased in cambium rings 1-4 of the taproot of the BvCPD overexpressing lines.In contrast, the activities of both enzymes decreased in the silenced lines, with the activity of SS decreasing more in the silenced lines and the decrease in R2 reaching a significant level across all rings.The enzyme activity of SS-C decreased to a lesser extent, with most of the differences being insignificant (Figure 2H,I).These results indicate that BvCPD increases sugar metabolism in sugar beet taproots.

BvCPD Can Improve Water and Nitrogen Uptake in Sugar Beets
Water and nitrogen uptake rates in sugar beets directly affect their growth.The water content, relative water content, and nitrogen accumulation in the taproots, petioles, and leaves of each BvCPD transgenic line were determined.The results showed that the water content of the BvCPD overexpression lines was higher than that of the WT in the taproots, petioles, and leaves, whereas that of the silenced lines was lower than that of the WT.The differences in water content among the transgenic lines in taproots were not significant.In leaves, except for the BvCPD overexpression line OE1, which was significantly higher than that of the WT, the differences between the transgenic lines and WT were not significant.Notably, in the petioles, the water content in the BvCPD overexpression lines ranged from 83.83% to 85.66%, which was significantly higher than that in the WT, whereas the water content in the silenced lines was lower than that in the WT but was not statistically significant (Figure 3A).Similarly, the relative water content in BvCPD overexpression lines surpassed that of the WT and silenced lines in taproots, petioles, and leaves, with particularly notable increases observed in petioles and leaves.Each BvCPD overexpression line demonstrated highly significant increases ranging from 6.28% to 8.98% in petioles and 3.20% to 6.12% in the leaves, while the silenced lines exhibited highly significant decreases ranging from 2.46% to 4.10% in the petioles and 4.98% to 6.99% in the leaves (Figure 3B).In addition, nitrogen accumulation in the taproots, petioles, and leaves of each BvCPD transgenic line was determined.Nitrogen accumulation in each organ was significantly increased in the BvCPD overexpression lines, whereas it was significantly or highly significantly decreased in the silenced lines (Figure 3C).These results indicate that BvCPD not only improves water uptake capacity but also significantly enhances nitrogen accumulation in sugar beets.

BvCPD Enhances the Activity of Enzymes Involved in Cellular Development
The activities of PAL and CAD were determined, and significant increases in PAL enzyme activity were observed in most cambium 1-4 ring layers of each BvCPD overexpression line taproot.Conversely, PAL activity showed significant decreases in silenced lines.The trend in the enzyme activities of CAD was also generally consistent across the transgenic lines, with a significant increase in the BvCPD overexpression lines and a decrease in the silenced lines; however, none of the decreases in cambium rings 1-3 rings were significant.Notably, the enzyme activities of CAD in the transgenic lines differed from those in the WT at highly significant levels in the 3-4 and 4 ring layers (Figure 4A,B).The combination of changes in the activities of the two enzymes indicated that the most significant changes in enzyme activities in lignin biosynthesis were found in the 3-4 and 4 ring layers, which had the greatest effect on lignin synthesis in sugar beet taproots.CesA, a key enzyme in cellulose synthesis, was significantly elevated in all BvCPD overexpression lines and significantly decreased in the silenced lines (Figure 4C).This suggests that BvCPD enhances cellulose synthase activity by promoting BR synthesis, which causes an increase in cellulose content, and consequently affects sugar beet taproot development.
In addition, PSP, a regulator of cell proliferation/differentiation, may be associated with changes in the parenchyma cells in cambium rings 1-4 of the taproot of BvCPD transgenic lines.The enzyme activity of PSP was measured, and the transgenic and WT lines showed a decreasing trend in enzyme activity from cambium ring 1 to ring 4.However, all BvCPD overexpression lines exhibited significantly higher enzyme activity than the WT across cambium rings 1-4, while all silenced lines displayed lower enzyme activity than WT.Significant differences were observed in cambium rings 1, 1-2, 2, and 2-3 of the silenced lines, whereas differences in cambium rings 3, 3-4, and 4 did not reach statistical significance (Figure 4D).The results showed that PSP significantly promoted cell division in cambium rings 1-4 rings of the taproot of BvCPD overexpression lines, but mainly interfered with the enzyme activities of the first three rings of the taproot in the silenced lines, thereby inhibiting the growth of sugar beet taproot cells.

BvCPD Regulates the Accumulation and Distribution of Dry Matter in Sugar Beet
The level of dry matter accumulation is directly related to growth conditions.The dry matter accumulation of taproots was greater than that of leaves and petioles across all lines.In the BvCPD overexpression lines, the dry matter accumulation in the taproots, petioles, and leaves was significantly higher than that in the WT by 14.99-10.54%,125.45-172.57%,and 31.74-50.56%,respectively.The dry matter accumulation of each organ of the silenced lines decreased significantly or highly significantly by 26.58-33.24%,48.41-56.85%,and 31.15-38.26%,respectively, compared with that of the WT.The total dry matter accumulation of the BvCPD overexpressing lines and silenced lines were also significantly higher and lower than that of the WT, respectively (Figure 5A).Moreover, the size relationship of the root-crown ratio followed the pattern of BvCPD overexpression lines < WT < silenced lines, with each transgenic line differing significantly from the WT (Figure 5B).

BvCPD Can Improve the "Source-Sink-Flow" Balance in Sugar Beets
The strength of crop "source" and "sink" along with the smoothness of "flow" play a decisive role in crop yield.When the physiological functions of the tissues and organs that produce and accept photosynthetic products are enhanced, crop yields increase, as has been demonstrated for crops such as maize [16], wheat [17], rice [18], potato [19], and cotton [20].In this study, BvCPD can improve the photosynthetic performance of sugar beet leaves by increasing photosynthetic pigments and enhancing photosystem II activity, strengthening the "source" of sugar beets.Higher chlorophyll content increases the rate of electron transfer during photosynthesis, which improves the efficiency of photosynthesis and allows plants to absorb and utilize light energy more efficiently.The absorption of light energy by chlorophyll prompts plants to produce more energy and electron-supplying substances such as adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) in photosynthesis, thus promoting the synthesis and accumulation of organic matter [21,22].Our research also confirmed this, with the overexpression of BvCPD resulting in a significant increase in dry matter accumulation in various organs and across the entire sugar beet plant, whereas silencing the expression of this gene led to a significant decrease in dry matter accumulation.Notably, the exogenous application of BRs in sugar beets during the rapid growth period of leaves and the period of root and sugar accumulation can increase the chlorophyll content, enhance the net photosynthetic rate and stomatal conductance of leaves, and strengthen photosynthesis, ultimately leading to an increase in yields [23].In our study, although the net photosynthetic rate did not show significant differences among the BvCPD transgenic lines, the leaf area index of the overexpressing line was significantly higher than those of the WT and silenced lines.This increase in leaf area index is sufficient to enhance overall photosynthesis in plants, resulting in increased production of organic matter and promoting an increase in the dry weights of the taproots, petioles, and leaves.
The CPD gene, as a key enzyme in BR synthesis, promotes the accumulation of BR, thereby influencing plant growth [12].BR can promote the growth of the cell wall and also promote the development of secondary xylem by regulating genes related to cell wall biosynthesis, such as BvXTH33, BvSHV3, BvCESA6, BvPARVUS, and BvCEL1 [3].
The BR biosynthesis mutant cpd, the BR receptor mutant bri1, and the BR signal transduction mutant bin2 produce fewer vascular bundles.Conversely, overexpression of BRI1 and BR signal transduction genes in plants results in more vascular bundles [24].BvCPD plays a positive role in BR synthesis in sugar beets, regulating the division and expansion of parenchyma cells in cambium rings 1-4 of sugar beet taproots.Additionally, it improves the development of the vascular bundle xylem and vessel lumens and leads to the accumulation of lignin and cellulose, ultimately promoting taproot enlargement [11].In the present study, we discovered that BvCPD improved the development of vascular bundles and parenchyma cells in the cambium of sugar beet taproots by increasing the activity of enzymes PAL, CAD, CesA, and PSP, thereby enhancing the absorption of water and nitrogen by sugar beets.This facilitates nutrient transportation in both the shoots and roots of sugar beets and increases the accumulation of dry matter in various organs.In essence, following BvCPD-mediated synthesis of BR, the yield of sugar beet taproots is enhanced by the expanded "sink" storage capacity, the photosynthetic characteristics of sugar beet leaves were improved, and the functionality of vascular tissues was enhanced.These changes lead to sufficient "source" provisioning and ensure the smooth "flow" within the plant system.

Regulation of Energy and Sugar Metabolism by BvCPD in Sugar Beets
Energy and sugar metabolism are important physiological processes in the growth and development of sugar beets and affect their yield and sugar content.Spraying BR during the early stages of sugar beet growth significantly promotes yield and sugar production.BvCPD can promote BR synthesis, enhance endogenous BR signaling, and regulate sugar beet development [11].In the present study, the main products of sugar metabolism were altered in each BvCPD transgenic line.The enzyme activities of SS and SS-C were significantly enhanced in the BvCPD overexpression lines.However, owing to the stronger activity of enzymes in the catabolic direction than in the synthesis direction, sucrose was decomposed into more fructose, resulting in a decrease in sucrose content, and consequently, a reduction in the accumulation of soluble total sugars.However, enhanced enzyme activity in the catabolic direction did not decompose more glucose, but rather its content decreased.Glucose catalyzes the formation of ATP via the glycolytic pathway to provide energy for life activities [25].Therefore, we speculated that the decrease in glucose was due to the production of more ATP through the glycolytic pathway to provide energy for sugar beet growth.The increase in ATP content in the BvCPD overexpression line taproots also supports this speculation.Furthermore, enhancement of the respiratory rate in the overexpression lines also releases energy stored in the form of ATP.Many glycolytic pathway intermediates provide carbon skeletons as raw materials for synthesizing other substances [26].PGK, a key enzyme in the glycolytic pathway, catalyzes the conversion of 1,3-bisphosphoglycerate (1,3-bPGA) and ADP to 3-phosphoglycerate (3PGA) and ATP while exerting reverse catalytic activity in gluconeogenesis and the Calvin cycle [27].We measured the activity of PGK in sugar beet taproots and found that its activity was enhanced in the BvCPD overexpression lines, thus validating our hypothesis.ZmPGKs are involved in photosynthetic metabolism, playing a major role in maize plant growth optimization [28].The interaction between poplar PtPGK, PtTPI, and PtGAPDHs is closely related to carbohydrate metabolism and is involved in plant development and stress resistance [29].ADH relies on NAD to catalyze the interconversion between ethanol and acetaldehyde, maintain glycolysis, and promote plant root growth [30][31][32].Exogenous application of BR significantly increases the level of ADH enzymatic activity in apples [33].In this study, we found that the accumulation of endogenous BR led to the accumulation of ADH in sugar beet taproots, thereby improving energy metabolism and promoting root development.Conversely, in the silenced lines, the decrease in SS activity led to a reduction in sucrose and soluble total sugar content, whereas SS-C activity showed no significant difference, resulting in no significant difference in the decomposed fructose content.However, the glucose content was lower than that in WT.Combining the findings that sugar beet taproots exhibited reduced respiration rate, ATP content, and PGK and ADH activities, we propose that the silenced lines may have experienced delayed growth and development, leading to decreased ATP synthesis compared to the WT.Consequently, less energy was consumed by the plant, resulting in glucose not being fully metabolized into ATP to support plant growth.This led to higher glucose content than in the WT.However, the specific molecular regulatory mechanisms require further investigation.Therefore, future research should focus on determining the mechanism of action of BvCPD in the growth of sugar beet taproot and identifying genes involved in physiological metabolism.

Conclusions
BvCPD can improve the photosynthetic characteristics of sugar beet leaves, increase the respiratory rate and ATP content of sugar beet taproots, and alter the sugar fraction content.These changes are associated with BvCPD-induced enhancements in the activities of PGK, ADH, SS, and SS-C.BvCPD also enhances water absorption and nitrogen accumulation in sugar beet plants by positively modulating the activity of PAL, CAD, CesA, and PSP.The regulatory effect of BvCPD on the physiological metabolism of sugar beets ultimately promotes an increase in plant dry matter accumulation.This study is of great theoretical significance for determining the regulatory mechanisms of BvCPD in sugar beet growth and offers valuable application information for the promotion of sugar beet yield improvements.

Figure 1 .
Figure 1.BvCPD increased energy metabolism in taproots by enhancing BR synthesis.(A) Differences in the respiration rates of taproots across different transgenic lines, (B) changes in ATP content of taproots across different transgenic lines, and (C,D) changes in the activities of PGK and AHD enzymes in cambium rings 1-4 of taproots in each transgenic line.* p < 0.05 and ** p < 0.01 by Student's t-test.

Figure 2 .
Figure 2. BvCPD regulates the sugar metabolism levels in sugar beets.(A) Sugar content, (B) taproot weight of a single plant, (C) sugar yield of a single plant, (D) total soluble sugar content, (E)

Figure 3 .
Figure 3. BvCPD improved water and nitrogen uptake by sugar beets.(A) Water content.(B) Relative water content.(C) Nitrogen accumulation in a single plant.* p < 0.05 and ** p < 0.01 by Student's t-test.

4 .
BvCPD enhanced the activity of cell growth-related enzymes.Analysis of the enzyme activities of (A) PAL, (B) CAD, (C) CesA, and (D) PSP in cambium rings 1-4 of the taproots of each transgenic line and WT.* p < 0.05 and ** p < 0.01 by Student's t-test.

Figure 5 .
Figure 5. BvCPD altered the dry weight and root-crown ratio in sugar beets.(A) Changes in the dry weight of taproots, petioles, and leaves of each transgenic sugar beet.(B) Changes in the rootcrown ratio of each transgenic sugar beet.* p < 0.05 and ** p < 0.01 by Student's t-test.

Table 1 .
Effect of the BvCPD gene on the function of sugar beet leaves.