Boron Ecient Sugar Beet (Beta Vulgaris L.) Variety Relieve The Symptoms of Boron Deciency By Enhancing The Antioxidation And Boron Utilization Capacity of The Root System

(Aims) Sugar beet is one of the most sensitive crops to boron and boron deciency inhibits the root growth and causes hollow symptoms in beets. However, how the roots of boron ecient sugar beet variety adapt to the morphology, physiological, and transcriptome mechanisms of boron deciency are rarely reported. (Method) Thus, the present study was carried out with B ecient sugar beet variety (H, KWS1197) and B inecient variety (L, KWS0143), and two B levels i.e., B0.1 (0.1 μM H 3 BO 3 , deciency) and B50 (50 μM H 3 BO 3 , control) were designed for hydroponic experiment. (Result) Boron deciency reduced the total root length, root forks, and root biomass of sugar beet. Compared with L variety, H variety have higher boron transport coecient, boron distribution ratio above ground, peroxidase and catalase activities, lower malondialdehyde content and reactive oxygen species accumulation. Transcriptome data showed that the two comparison groups, HB0.1 vs HB50 and LB0.1 vs LB50, were enriched for 537 and 257 differentially expressed genes, respectively. The H variety mainly induced and regulated the GO term enrichment associated with antioxidant and stress resistance. On the contrary, the L variety induced cell death and negative regulation of biological and metabolic processes. (Conclusion) B ecient variety specically up-regulated boron deciency response genes to activate the antioxidant enzyme system, promoted rational root conguration, and enhance plant growth antioxidation and resistance to boron deciency. The results of this study serve as a theoretical basis of screening candidate genes that respond to boron deciency and adaptation mechanism of boron deciency. can and Boron signicantly Compared with normal boron signicantly the and CAT enzyme activities of H oxidative damage of H The H variety also has a strong boron utilization ability, and can accumulate more boron nutrition under the condition of boron deciency. At the same time, this study also showed that ecient boron variety have complex transcriptional molecular network changes that regulate the up-regulation of antioxidant activity, peroxide and oxidoreductase activity and other related Secondly, the H variety also regulated the expression of antioxidant enzymes, transcription factors and other boron deciency-responsive genes. In short, the ecient B variety enhanced the plant's antioxidant capacity by regulating the expression of boron deciency response improved the boron utilization eciency, and alleviated the symptoms of boron deciency in the

Introduction 2010; Huang et al., 2021). Generally, B was absorbed by plants in the form of boric acid (H 3 BO 3 ) through passive diffusion (Raven, 1980;Dordas and Brown, 2000). However, in the absence of B, plants transport boron from soil to roots by inducing aquaporins (NIPs, nodulin26-like intrinsic protein (Roberts and Rouray, 2017; Granado, 2020) and the BOR family of borate transporters, then load B into the xylem for further transportation.
Transcriptomics was widely used to study plant responses to various stresses (Imadi et al., 2015), which can obtain differentially expressed genes (DEGs) based on changes in genotype or growth environment between species (Mazzitelli et al. 2017). In recent years, more and more studies have used RNA sequencing (RNA-seq) to reveal the molecular response mechanism of a variety of crops under abiotic stress (Jiang, et al., 2020). The publication of the sequencing of the sugar beet genome makes sugar beet an excellent model crop for studying plant responses to various stresses (Dohm et al. 2014). At present, it has been reported that sugar beets have been exposed to salt stress (Yu et

Experimental design, plant material, and treatments
The experiment was carried out in the arti cial light cultivation room of the National Sugar Improvement Center (Harbin, China) in August 2019, located (longitude: 126°37′, latitude: 45°43′). The test material had boron e cient variety "KWS1197" (H) and boron ine cient sugar beet variety "KWS0143" (L) purchased from KWS company (KWS SAAT SE & Co. KGaA, Germany). Before sowing, rinse the seeds in running water for 6h, then transfer to 75% alcohol to rinse for 1min, and nally transfer to 2‰ of thiram solution to soak for 12h, then washed with distilled water 3 times, and nally sown in high-temperature sterilized vermiculite.
The seedlings with the same growing vigor were selected when the cotyledons of sugar beet are fully expanded for the hydroponic experiment. The 2L polyethylene plastic nursery pot (covered with black 2.3 Antioxidant enzyme system index determination and histochemical staining of root system The 0.5 g of fresh root samples were homogenized in 5 ml of 0.05 M phosphate-buffered saline (PBS) at pH 7.8 to a pre-cooled mortar (sampling process is the same as the transcriptome) into a homogenate and centrifuged at 15000 rpm at 4°C for 15 min. The supernatant was stored in a refrigerator at 4°C for testing.
Nitrogen blue tetrazolium reduction method (Beauchamp and Fridovich, 1971) to measure the SOD enzyme activity. The total reaction system had 3 ml mixture, and the OD value of 50 µl enzyme solutions were measured at 560 nm to calculate the SOD enzyme activity. The guaiacol method (Cakmak et al., 1991) was used to measure the OD value of 50 µl enzyme solutions at 470 nm, the change rate within 210 s was record, and calculated the POD enzyme activity. According to the method described by De Azevedo Neto (De Azevedo Neto et al., 2006), 50 µl enzyme solutions were measured at 240 nm and the OD value change rate in 210s was recorded to calculate the CAT enzyme activity. Vos (Vos et al., 1991) method was referred to prepare 4 ml total reaction system which had 1.5 mL of the enzyme solutions in a glass test tube with a stopper, and 2.5 mL 0.5% thiobarbituric acid (TBA) solution. After mixing, the samples were reacted in a boiling water bath for 20 min, and then the samples were cooled to room temperature and centrifuged at 15000 rpm at 4°C for 15 minutes. The OD values were recorded at the wavelengths of 532 nm, 600 nm, and 450 nm, and calculate the content of malondialdehyde (MDA) according to the formula.
Note: V is the total enzyme volume of 5 ml, and V t is the volume of 1.5 ml enzyme solution used to determine the content of malondialdehyde.

Determination of boron content in sugar beet plants
Boron content in plants was determined by curcumin colorimetry (Dible et al., 1954). The 0.2 g of dry samples (DW) of shoots and roots ground were ground to a uniform powder (passed through a 40-mesh sieve), and put in a mu e furnace to carbonize at 240℃ for 1 h, then adjust the temperature of the mu e furnace to 560℃, continued ashing for 4 h. The B was extracted with 10 mL of 0.1 M HCl for 30 min and ltered it as the test solution. The 1 ml of the test solution was pipetted into an evaporating dish, and 5 ml of curcumin-oxalic acid solution added and, shaked well and placed gently in a 54℃ water bath to evaporate to dryness, and then continued heating for 20 min (total evaporation time was about 45 min). The 10 mL of 95% ethanol was used to wash the evaporating dish several times in small amounts, ltered, and measured the OD at 540 nm by double-beam UV-spectrophotometer (UV-8000A, Shanghai, China). The 95% ethanol was used as a blank. The calculation formulae are as following: Boron accumulation (µg plant -1 ) = B content (µg g -1 ) × corresponding dry weight (g plant -1 ) Boron transport coe cient = aboveground B accumulation (µg plant -1 )/underground B accumulation (µg plant -1 ) Boron use e ciency (g µg -1 ) = plant dry weight (g plant -1 ) / plant boron accumulation (µg plant -1 )

Enrichment analysis of DEGs, GO terms and KEGG categories
In order to screen and enrich the differential expressedgene(DEGs) in response to boron de ciency, two comparison groups were constructed, namely HB0.

qRT-PCR veri cation of DEGs in the transcriptome
In order to verify the accuracy of the RNA-Seq data, 14 randomly selected DEGs were analyzed by qRT-PCR. Primer Express 5.0 (Premier Biosoft Interpairs, Palo Alto, CA) was used to design primers. The sequence of the primers was listed in Table S1. Plant Polysaccharide Polyphenol Extraction Kit (DP441, Tiangen, Beijing, China) was used to extract total RNA from root samples, and FastKing one-step method was used to remove genomic cDNA rst-strand synthesis premix reagent, the cassette (KR118-02, Tiangen, Beijing, China) performs reverse transcription to synthesize the rst-strand cDNA. The PCR products were detected by gel electrophoresis to check the speci city of gene primers. The relative expression levels of genes were detected by the Mx3000P real-time PCR system (Angelent, La Jolla, CA, USA) instrument and the Super Real PreMix Plus (Beijing Tianjian, China) kit was used for the qRT-PCR test. Each cDNA sample was performed three technical replicates. The 20 µL qPCR reaction system contained 1 µL cDNA, 1.2 µL primer pair mixture (10 µM), 0.4 µL 50× reference dye, 7.4 µL RNase-free ddH2O and 10 µL 2× SuperReal PreMix Plus. The qPCR reaction program using the SYBR Green I method is as follows: initial denaturation at 95°C for 10 minutes, then 40 cycles: 95°C for 10 s, 60°C for 20 s and 72°C for 30 s, followed by a melting curve analysis and measured whether the expression level was normal according to the expression of the housekeeping gene GAPDH, and checked the speci city of the ampli ed fragment according to the generated melting curve. The 2 −ΔΔCt method was used to analyze the relative expression of genes (Livak and Schmittgen 2001).

Statistical Analysis
SPSS 22.0 (SPSS Inc, Chicago, IL) software was used to conduct two-factor random analysis of variance (two-way ANOVA) and minimum signi cant difference method (LSD) at p < 0.05. Origin 2019b (origin 2019b Inc, USA) was used to draw the bar chart, line chart, and the level of signi cance was set to p = 0.05 or p = 0.01. Signi cant and extremely signi cant differences were expressed by "*" and "**". Figure 1A showed the growth of plants after 7 days of boron de ciency, the results showed that boron de ciency signi cantly inhibited the normal growth of sugar beet, which was manifested by the reduction of root elongation and root forks and promoted the occurrence of lateral roots (Fig. 1B), and as the stress time increased, the difference between the boron treatments was more obvious. Boron de ciency led to a decrease in the total root length (Fig. 1C), root volume ( Fig. 1D), and number of root tips (Fig. 1E) of the plant root system, increasing the average root diameter (Fig. 1F). Further analysis of the root scan data showed that boron de ciency signi cantly reduced the root neness (RF) of the plant (Fig. S2B), and the H and L varieties were reduced by 38% and 62%, respectively. Boron de ciency reduced the root length ratio (RLR), root mass ratio (RMR), root tissue density (RTD), and speci c root length (SRL) of sugar beet plants ( Fig. S2A, 2C, 2D, 2E), resulting in a decrease of 28.0%, 12.0%, 22.0% and 21% in H variety, respectively; but increased the speci c root surface area (SRA) of L variety (27.8%), while the speci c root surface area of H variety hardly changed (Fig. S2F).

Effect of boron de ciency treatment on the growth characteristics and biomass of sugar beet
As shown in Fig. 2, boron de ciency signi cantly reduced the root dry weight (25.03% and 34.39%) and root-shoot ratio (24.91% and 22.53%) of H and L variety, and boron de ciency also signi cantly reduced the shoot dry weight (14.98%) and total plant dry weight (17.96%) of L variety, while the shoot dry weight (0.66%) and total plant dry weight (4.76%) of H variety were not signi cant. The results showed that under the treatment of boron de ciency, the above-ground dry weight and total plant dry weight of H and L varieties showed extremely signi cant differences. However, the root dry weight and root-shoot ratio of the two sugar beet varieties showed no signi cant difference.
3.2 Responses of the antioxidant enzyme system in the roots of different boron-e ciency sugar beet cultivars to boron de ciency Boron de ciency signi cantly increased the POD (21%) and CAT (69%) enzyme activities of H variety and decreased the SOD enzyme activity (0.5%) ( Fig. 3A-3C). Boron signi cantly reduced the enzyme activities of SOD (12%), POD (19%) and of L varieties, and extremely signi cantly reduces the enzyme activity of CAT (74%). Under the two-boron treatments, the MDA content in the roots of the H variety was signi cantly lower than that of the L variety (Fig. 3D) variety was lower than that of the L variety, which indicated that with a small amount of dry matter, the H variety could induce more boron accumulation. Further analysis of the boron distribution rate in plant roots and shoots showed that in the case of boron de ciency, H varieties can transport more boron nutrients to the ground (Fig. 5F).

Overview of transcriptome data by RNA-Seq analysis
By using PE150 sequencing strategy, a total of 563551518 raw data were obtained, and 556750800 net readings and 10467 single genes were obtained after quality control. After removing the joint contamination and low-quality Reads, more than 21398452 valid original readings were obtained from H and L variety, and high-quality readings exceeding 21138827 were obtained for each treatment (Table 1); GC content was distributed in 92.49% and 92.61%. Among them, the ratio of Q20 was above 97.26%, and the ratio of Q30 was above 42.60%. All treatments had high-quality readings of more than 93.31%, which can be used to further analyze the transcriptome of sugar beet roots under boron de ciency stress.

Identi cation of differentially expressed genes
Two comparison groups were constructed to analyze the transcriptome data, and it was found that 537 and 257 DEGs existed in the HB0.1 vs HB50 and LB0.1 vs LB50 comparison groups, respectively (Fig. 5).
The results showed that there were signi cant differences in gene expression levels between the two varieties between normal boron control treatment and boron de ciency treatment (Fig. 6)

Enrichment analysis of GO term and KEGG pathway
Through GO term functional enrichment analysis, 288 differentially expressed GO terms were found in the HB0.1 vs HB50 comparison group. The results showed "response to oxidative stress" (GO:0006979, 3 upregulated and 3 down-regulated genes), "peroxidase activity" (GO:0004601, 3 up-regulated and 3 downregulated genes) and "thylakoid light-harvesting complex" (GO:0009503, 1 up-regulated gene) were the most signi cantly enriched GO among biological process (BP), molecular function(MF) and cellular component(CC). In the LB0.1 vs LB50 comparison group, 142 differentially expressed GO terms were found, "apoptotic process" (GO:0006915, 11 up-regulated genes and 1 down-regulated gene), "obsolete toxin activity" (GO:0015070, 4 up-regulated genes and 3 down-regulated genes) and "transcription factor TFIID complex" (GO:0005669, 4 down-regulated genes) were the most abundant GO techniques in BP, MF and CC ontology, indicating that the genes involved in these processes may be missing which play a key role in the response to boron stress. Interestingly, in the two comparison groups, neither the up-regulated expression of DEGs involved in cell components was enriched, and only partially down-regulated DEGs were enriched in the L variety. Secondly, the GO categories such as antioxidant activity, peroxide and oxidoreductase activity in the HB0.1 vs HB50 comparison group showed an up-regulated trend, while the LB0.1 vs LB50 comparison group had negative effects on cell processes, biological processes, and metabolic processes; and up-regulated cell death and programmed cell death GO categories. The adjustment process showed an upward trend, which may be related to the stronger antioxidant capacity of H variety.
In order to further analyze the functions of DEGs involved in the response to boron de ciency, the KEGG database was used to screen the differentially expressed genes enriched in different metabolic pathways.
In the KEGG pathway analysis, the enriched DEGs in the HB0.1 vs HB50 and LB0.1 vs LB50 comparison groups were divided into 68 and 47 functional categories, respectively (p < 0.05 as the screening threshold for KEGG functional enrichment). The study found that the number of DEG involved in the metabolic pathway was the highest (bvg01100), 50 and 25 DEG in the two comparison groups, respectively. Phenylpropanoid biosynthesis (14 DEGs) and fructose and mannose metabolism (3 DEGs) were the signi cantly enriched KEGG pathways in the two comparison groups HB0.1 vs HB50 and LB0.1 vs LB50, respectively.

Responses of antioxidant enzyme-related genes in sugar beet roots to boron de ciency stress
The results of the expression of candidate genes related to antioxidant enzymes involved in the response to boron de ciency revealed that in the H variety, there were 6 up-regulated genes and 1 down-regulated gene (Fig. 9). The expression of SOD-related genes (LOC104902979) was highly down-regulated between 3.8 Response of transcription factor-related genes to boron de ciency The study found that candidate genes related to transcription factor families, such as bHLH, WRKY, bZIP, MYB and B3 were differentially regulated between the two comparison groups (Fig.10), transcription factor-related candidate genes showed an up-regulated expression trend in H variety. For example, transcription factor bHLH01-like (LOC104889601) and transcription factor bHLH18-like (LOC104897819) were highly up-regulated in the HB0.

Response of candidate genes related to aquaporin and boron transporter to boron de ciency
The results showed that the candidate genes of aquaporin in sugar beet were differentially induced and regulated (Fig. 11). Aquaporin gene NIP5-1 (LOC104895986 and LOC104895985) were highly upregulated in the two comparison groups. Compared with B de ciency treatment, the gene expression levels were 1.980 and 1.099, respectively, while LOC104893298 was highly downregulated in both groups Regulated, the expression levels are − 5.072 and − 1.161. Aquaporin gene NIP6-1 (LOC104904889), boron transporter BOR2 (LOC104894816) and boron transporter 4 were up-regulated in the two varieties, and NIP7-1 (LOC104906876) was down-regulated in the two comparison groups. Compared with boron de ciency treatment, the gene expression level was − 1.767 and − 0.035. Probable boron transporter 7 (LOC104887698) was highly down-regulated in the H variety, and the gene expression level was − 2.544.

qRT-PCR validates transcriptome data
In order to verify the accuracy and reproducibility of the Illumina RNA-seq results, 14 representative genes such as those involved in cell wall synthesis, POD enzymes, carbon and nitrogen metabolism and other related genes were selected to verify these by real-time quantitative PCR (qRT-PCR). The relative expression level of the selected gene was further compared with the relative expression from RNA-seq analysis. Although the results of qRT-PCR were not exactly the same as the results of RNA-seq, the qRT-PCR results showed that the relative gene expression trends were consistent with the RNA-seq data, which veri ed the accuracy of the transcriptome sequencing results, thus supporting the reliability of the RNAseq results in this study. . Consistent with the law described above, in this study, boron de ciency reduced the total root length, root volume and root forks of sugar beet. Moreover, boron de ciency signi cantly reduced the biomass of sugar beet, such as root fresh weight and root dry weight ( Fig. 2A; Fig. 2C). Further analysis of the root morphology and growth indicators of sugar beet showed that boron de ciency signi cantly reduced the root neness (RFs) of H variety (52%) and L variety (46%) (Fig. S2B). Boron de ciency also reduced root length ratio (28%), root mass ratio (12%), speci c root length (21%) and speci c surface area (0.1%), but the reduction of H variety was less than that of L variety (Fig. S2)  was manifested in the signi cant increase of the POD and CAT enzyme activities in the root system under the boron de ciency (Fig. 3B, 3C). Regardless of the treatment, MDA content was reduced in the root system (Fig. 3D). In addition, through enrichment analysis of DEGs, it was found that H variety maintained the regularity and order in the roots of H variety by regulating the up-regulated expression of antioxidant enzyme-related genes such as SOD (LOC104899994), POD (LOC104908142) and CAT (LOC104905646) candidate genes (Fig. 9). Boron e cient variety reduced the MDA content and ROS accumulation in roots by regulating the synergy between antioxidant enzyme systems (Fig. 4), assisting plants in resisting stress and reducing plant oxidative damage.

Boron e cient sugar beet variety promotes the e cient absorption and transport of boron by inducing the expression of aquaporins and boron transporters
Plants control the absorption and transport of boron by regulating two different types of boron transporters, namely the borate channel of the main intrinsic protein family and the borate transporter of the BOR family . Studies have proposed that NIP7;1 serves as a gated boric acid channel in developing anthers that aids in the uptake of this critical micronutrient by tapetal cells (Routray, 2018).
Under B restriction conditions, BOR2 can transport boric acid/borates in roots from symplasts to apoplasts, and helps RG-II to effectively cross-link in the cell wall and promote root cell elongation (Miwa, 2013). Under high boron treatment, plants can reduce the B concentration in roots through BOR4 mediated B e ux, maintain plant boron steady state, and increase plant tolerance to high boron environment (Miwa, 2011). In our research results, NIP7-1 (LOC104906876) and BOR7 (LOC104887698) showed a downward-regulated expression trend in the H variety, while BOR2 (L0C104894816) and BOR4 (LOC104904614) showed an up-regulated expression trend in the H variety, indicating that the H variety may It also regulates the expression of the above genes, reduces the e ux of boron, and promotes the elongation of plant roots.
At low boron concentrations, plants induced the expression of NIP5-1 to complete the initial boron uptake  and promoted e cient boron absorption and boron nutrient accumulation by roots (Wang et al., 2017). BOR1 was the rst boron transporter to be identi ed (Miwa, 2006), and it is mainly involved in the loading process of xylem in roots under low boron conditions (Larson, 2019). In this study, the relative expression levels of aquaporin and boron transfer genes in sugar beet roots were analyzed by gene uorescence quantitative PCR, and it was found that the expression of NIP5-1 (LOC104895985, LOC104895986), NIP6-1 (LOC104904889), and BOR2 (LOC104894816) were up-regulated in H variety, and the relative expression of genes was higher than that in L cultivars (Fig. S3). Compared with the L variety, the H variety also had higher boron absorption and transfer capacity ( Fig. 5C; Fig. 5D In addition, through the construction of e cient physiological and molecular patterns of boron e cient genotypes (Fig. 13), further analysis and comparison of the root morphology of different boron-e cient beet varieties con rmed these conjectures and inferences, overall, under the induction of boron de ciency, the boron-e cient variety activated the physiological defense response, maintained the regular antioxidant enzyme system, reduced the MDA content and the accumulation of active oxygen in the root system, and reduced the oxidative damage of the plant. In a word, boron-e cient varieties also upregulated the expression of related candidate genes such as TFs, transporters and hormones to establish a molecular interaction network system for e cient boron absorption and utilization to alleviate the symptoms of plant de ciency.

Conclusions
The results showed that boron de ciency can inhibit root elongation and reduce root-to-shoot ratio and root fork, thereby affecting the normal growth of roots.       The response of antioxidant enzyme-related genes to boron de ciency stress. The heat map shows the differential regulation of antioxidant enzymes such as SOD, POD and CAT in sugar beet varieties with different boron e ciency under boron de ciency. The red in the gure indicates that the gene is upregulated, and the blue indicates that the gene is down-regulated. The speci c data indicates the relative expression of the gene Log2(fold change) (Log2(FC)).

Figure 10
The effect of boron de ciency on the expression of transcription factor-related genes. The heat map showed the differential regulation of transcription factors such as bHLH, WRKY, bZIP, MYB and B3 and Page 32/34 other related genes in sugar beet varieties with different boron e ciency under boron de ciency. The scale color represents Log2(FC), the red in the gure indicates that the gene is up-regulated, and the blue indicates that the gene is down-regulated.

Figure 11
The effect of boron de ciency on the expression of genes related to boron absorption and transporter.
The heat map shows the differential regulation of Candidate genes related to boron uptake and transport such as NIP5-1, NIP6-1, NIP7-1, BOR2, BOR4 and BOR7 in sugar beet varieties with different boron e ciency under boron de ciency. The scale color represents Log2(FC), the red in the gure indicates that the gene is up-regulated, and the blue indicates that the gene is down-regulated.  Schematic diagram of physiological and molecular mechanisms of e cient use of boron e cient variety.
The seedlings of sugar beet varieties with different boron e ciency were cultivated in boric acid nutrient solution containing 0.1 μM (boron de ciency) and 50 μM (normal boron). The gure shows the difference in root morphology after 7 days of boron de ciency. It can be seen from the gure that the stress of boron de ciency leads to the disorder of the antioxidant system, which increases the MDA content and the accumulation of ROS in the root system, which in turn leads to oxidative damage and inhibits root elongation and normal growth. Boron e cient beet variety maintains a regular antioxidant system and regulates the up-regulated expression of related candidate genes such as transcription factors, transporters, boron transporters, and hormones, thereby increasing the ability of plants to absorb and transport boron and alleviate the symptoms of boron de ciency in beets.

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