Screening and Identification of Cinnamoyl CoA Reductase Genes Associated with Lignin Synthesis and Abiotic Stress in Boehmeria nivea

ABSTRACT Ramie (Boehmeria nivea (L.) Gaudich.) is a bast fiber crop that can be used in textiles, medicine, and feed and for environmental protection. Lignin plays an important role in plant defense and development, but lignin is a critical factor affecting the fiber and feed quality of ramie. Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin biosynthesis that catalyzes the synthesis of these common precursors. Here, eight CCR genes of ramie were identified based on sequence characteristics, gene structure, phylogenetic relationships, stress response to abiotic treatment, and the correlation of lignin and flavonoid contents with the expression level of genes. The eight CCR genes were cloned and divided into three groups with significant differences in their structures and sequence characteristics. BnCCR1 and BnCCR5 are involved in lignin biosynthesis. BnCCR4 plays a role in defense-related processes. It is worth noting that BnCCR2 is different from the structure and function of typical CCR, which is closely related to the synthesis of both lignin and flavonoids, and may play different roles in the xylem and phloem of ramie. The functions of the other genes require further investigation. These findings provide a basis for subsequent functional verification of CCR genes using genetic engineering.


Introduction
Ramie (Boehmeria nivea (L.) Gaudich.) is a natural and renewable phloem fiber crop that can be harvested in three to four seasons per year.It has been studied for use in textiles, medicine, feed, and environmental protection (National hemp industry technology system 2017; Rehman et al. 2019;Xu et al. 2021).However, the application of ramie is limited to the traditional textile field, and the depth of its integration with other related industries is insufficient, which limits its efficient application of ramie (Rehman et al. 2019).In recent years, ramie has been planted as one of the preferred crops for remediation of soil contaminated by heavy metal pollution in southern China, and combined with the traditional advantages of raw material production, textile processing, brand building, and scientific and technological support, good social and economic benefits have been achieved (Chen, Zhu, and Xiong 2020;Wu et al. 2022).Therefore, to use fiber, feed, environmental protection, and other diversified product structures to effectively deal with the risks brought by changes in the traditional single product market, cultivation of new varieties of ramie with multiple functions such as excellent fiber quality, high nutritional value, and strong stress resistance is necessary.
Lignin is a vital biomass component in the vascular tissues of plants with various functions, including acting as mechanical support or a barrier against pathogens and diseases (Guo et al. 2020;Zhao 2016).However, lignin is also a critical factor affecting the quality and nutritional value of ramie bast fiber.The cultivation of excellent ramie varieties with low lignin content in phloem fibers, feeding, and ensuring strong stress resistance have become scientific problems to be solved urgently.The H, G, and S units are polymerized into lignin (Zhao et al. 2021).The ratio of S, G, and H units in ramie lignin is approximately 6:3:1 (Wei 2016), which is different from that in the model plant.The G unit is 2-3 times more abundant than the S unit in Arabidopsis (Berthet et al. 2011).The lignin in poplar consists mainly of G units, with a minor amount of H units (Yan et al. 2019).The intermediate substances from the metabolism of phenylpropane, such as cinnamoyl CoA and coumaroyl CoA, are catalyzed and then undergo different pathways to form the lignin monomer under the catalysis of enzymes such as CCR (Figure 1) (Ni 2019).Enzymes and their corresponding genes associated with the metabolic pathways leading to the production of this complex phenolic polymer have been studied for many years and are relatively well characterized.The use of genetically modified model plants (Arabidopsis, tobacco, poplar) and mutants has contributed greatly to our current understanding of this process (Neutelings 2011).In this context, the natural hypolignification of secondary cell walls in plant bast fiber species such as hemp ramie is starting to provide novel information about how plants control secondary cell wall formation (Neutelings 2011).However, most of the research on ramie lignin has focused on the degradation and removal of lignin from the fiber (Liu et al. 0000;Zhang et al. 2023), and there are few studies on lignin biosynthesis.The structure and content of phloem fiber lignin differ significantly from those of typical woody plants.This suggests that phloem fiber crops represent a new perspective for studying unusual lignification processes.
CCR (EC 1.2.1.44)was validated as the first committed enzyme in the monolignol-specific biosynthetic pathway.CCR catalyzes reduction reactions using various cinnamoyl-CoA esters, such as cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA, to produce their corresponding hydroxycinnamaldehydes, which are further converted into different lignin monomers by another reductase, cinnamyl alcohol dehydrogenase (Figure 1) (Chao et al. 2019).CCR gene families are widely distributed in the plant kingdom, and CCR-like genes are found in ferns, gymnosperms, angiosperms, and some photosynthetic algae.Different CCRs differ in their catalytic efficiency and preference for the substrate, such as in Arabidopsis thaliana AtCCR1 (Baltas et al. 2005), and in Triticum aestivum TaCCR1 and TaCCR2 (Ma 2007), all of which show substrate preference for feruloyl-CoA.In Panicum virgatum (Escamilla-Trevino et al. 2010) and Medicago truncatula (Zhou et al. 2010), PvCCR1 and MtCCR1 preferred feruloyl-CoA, whereas PvCCR2 and MtCCR2 preferred caffeoyl-CoA and coumaroyl-CoA.This was consistent with the involvement of CCR in lignin biosynthesis.Moreover, flavonoid biosynthesis starts with phenylpropane metabolism, and its products, cinnamoyl-CoA, coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, are catalyzed by CHS, CHI, and F3H to form chalcones (Figure 1).Limiting carbon flow down the monolignol pathway should enhance the availability of coumaroyl CoA esters, which are some of the substrates of chalcone synthase, the first catalytic step toward flavonoid synthesis (Anterola and Lewis 2002).Transgenic plants exhibiting reduced lignin content were obtained using an RNAi strategy targeting tomato CCR1, and the vegetative organs of the CCR1 down-regulated plants contained higher amounts of chlorogenic acid and rutin (van der Rest et al. 2006).
In addition, functional studies of CCR family members in Arabidopsis thaliana revealed that AtCCR1 is involved in lignification, whereas AtCCR2 is mainly induced by biotic stresses and participates in plant defense mechanisms (Lauvergeat et al. 2001).In switchgrass, PvCCR1 and PvCCR2 were found to possess CCR activity; PvCCR1 might function in lignin biosynthesis during the developmental stage, and PvCCR2 might be involved in stress defense (Escamilla-Trevino et al. 2010).Overexpression of CCR2 in brassica napus increases its resistance to S. sclerotiorum by affecting lignin biosynthesis (Liu et al. 2021).However, whether CCR genes are also involved in plant defense mechanisms in ramie has not yet been investigated.Therefore, screening and identifying CCR genes associated with ramie abiotic stress is essential.
The whole genome of ramie has been released, making it possible to perform a genomewide analysis of the CCR gene family and conduct validation experiments (Chen et al. 2023;Liu et al. 2018;Luan et al. 2018;Wang et al. 2021).In this study, we searched the Ramie genome database and identified eight CCR family members.We further performed systematic analyses of gene structures, primary structures, conserved motifs, phylogenetic relationships, and three-dimensional structures.Finally, we cloned and further analyzed their responses to abiotic treatments and the correlation of lignin and flavonoid contents with the expression level of genes in the phloem and xylem of ramie stems at different periods to explore the BnCCRs protein structure and possible function in lignin biosynthesis.Genuine CCRs from ramie were screened and identified, providing a basis for subsequent functional verification using genetic engineering.Therefore, it is essential to breed new ramie varieties with multiple functions.

Plant materials and sample preparation
Ramies were planted in the field for stump propagation and routine management.The stems of wild ramie in the seedling period (stem full cyan), vigorous growing period (stem black rod 1/3), and mature period (whole stem black rod) were selected as study materials.Six plants randomly selected with the same growth tendencies were cut into small pieces of 1 cm.For these pieces, the bark (phloem) and rod (xylem) of the stem section were separated using a method described by Tang et al. (2019) and quickly frozen in liquid nitrogen.After mixing the six samples, samples were evenly stored in a refrigerator at − 80°C for the extraction of total RNA followed by qRT-PCR, and the remaining sample materials were dried in an oven at 60°C and ground to a powder in a tissue grinding mill MM 400 (RETSCH, Haan, Germany).The powder was sieved through a 30-mesh screen to determine the lignin content and flavonoid content.Lignin and flavonoid contents were determined using a Lignin Content Assay Kit and Flavonoid Content Assay Kit (Sangon, Shanghai, China), respectively, according to the manufacturer's protocol.Mature stems were selected for total RNA extraction from the gene clone.

Abiotic stress treatment
The ramie seedlings were cut according to the method described by Li et al (Li 2017).Seedlings with similar growth conditions (~12-15 cm high) were used for the hydroponics.Excess leaves were removed and 2-3 functional leaves at the top were left.The bottom of the stem was removed, and the length of the seedlings was consistent to ensure the uniformity of the test materials.The treated seedlings were sterilized for 30 s using the 500 × carbendazim solution, fixed in the foam plate hole of the hydroponics apparatus with a sponge, and placed in a hydroponic box such that the bottom of the cuttings exceeded the foam plate by approximately 2 cm to ensure that the cuttings were completely in contact with water.Forty seedlings were placed in each pot in a hydroponic room for cultivation.The light dark cycle was set to 16/8 h, and the room temperature was controlled at 25-30°C.After 3-4 days, young roots of the cut seedlings first appeared.Nutrient solutions A and B were added according to the manufacturer's instructions, and cultivation was continued.During this period, the nutrient solution was changed every 7 days.When the average plant height of the ramie seedlings reached 20-22 cm, the seedlings with consistent growth were randomly divided into three groups, and 10 seedlings in each group were used for treatments under different abiotic stresses.The nutrient solution was replaced, CdCl 2 was added for treatment, the Cd 2+ concentrations of each basin solution were 0, 5, 10, 20, and 40 mg/L, and the pH was adjusted to 6.0 with phosphoric acid.The seedlings were also treated with CdCl 2 (48 h).The harvested ramies were first soaked in deionized water for 15 min to remove Cd 2+ attached to the root surface.Three seedlings were randomly selected for each treatment group, and the stem segments were mixed and flash-frozen in liquid nitrogen and then stored in a refrigerator at − 80°C until total RNA extraction.Three replicates were performed for each treatment group.NaCl was added for treatment, and the concentration of each basin solution was 0, 0.8, 1.6, and 2.0 g/L, respectively; the remaining steps were performed as described above.

Total RNA extraction and cDNA synthesis
Total RNA from the stem, phloem, and xylem of ramie was extracted using a FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China).The concentration of total RNA was measured using a Thermo Fisher Scientific Oy spectrophotometer 1510 microplate reader, and the integrity of total RNA was determined by 1.0% agarose gel electrophoresis.A HiScript® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) was used to synthesize cDNA for PCR and qRT-PCR.After the quality of cDNA was tested using primers for the reference gene Actin I and the cDNA concentration was determined by Thermo Fisher Scientific Oy spectrophotometer 1510 microplate reader, the samples were stored at − 80°C until further analysis.

Identification and gene structure analysis of CCR genes in the ramie genome
Ramie reference genome data (GCA_0181312145.1)(Wang et al. 2021) were obtained from NCBI.The ramie genome sequences were analyzed by comparative alignment based on nucleotide sequences of three CCR genes (Tang et al. 2022) that have verified their biochemical function by locality BLAST, and redundant sequences were manually removed (Insert Table S1 here).These genes were screened and renamed based on their orthologous relationship with AtCCR, their relative position on the ramie chromosome, and the results obtained using BLASTP alignment with the SwissProt database.Eight CCR candidate ramie CCR genes have been identified.The corresponding CCR locations, coding sequences (CDS), introns, and untranslated regions (UTR) were obtained from the annotation file of the ramie genome.

Cloning of CCR genes in ramie
Primer 5.0 was used to design PCR primers based on the CDS of genes from the genome in ramie, and primers were designed and synthesized Insert (Table S2 here).The PCR system according to the method described by Tang et al (Tang et al. 2018).The PCR products were purified from a 1% agarose gel using the FastPure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China) and cloned into the pNC-AEnTopo vector (NC Biotech, Hainan, China).The inserts were sequenced for confirmation (Tsingke Biotechnology Co., Ltd., Beijing, China).

Expression profile analysis of CCR genes in ramie
Primer 5.0 was used to design the qRT-PCR primers (Insert Table S4 here) within the conserved domain database sequence regions based on the nucleotide sequences of the CCR genes.The qRT-PCR reaction system contained 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.5 μL of cDNA, 0.4 μL of Primer-P1, and 0.4 μL of Primer-P2, sterile water was added to ensure a total volume of 20 μL.The amplification reaction program was as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.The melting curve program was as follows: 95°C for 15 s, 60°C for 60 s and 95°C for 15 s using LightCycler 480 Software release 1.5.0 (Roche, Basel, Switzerland).Three biological replicates were performed, and the sum of all copies of CCR genes in the stem, phloem, and xylem was determined.Considering Actin I as the reference gene, the expression level of each gene was determined by analyzing data through the 2 −ΔΔCt method.

Statistical analysis
All experiments were independently conducted at least three times.Statistical significance analysis of all data was performed using SPSS software (version 26.0; SPSS, Chicago, IL, USA), with one-way ANOVA and Duncan's multiple range test.Subsequently, gene expression levels, lignin content, and flavonoid content were analyzed with bivariate correlations using SPSS software (version 26.0).The results of the one-way ANOVA are labeled using letters.Other significance was designated as follows: *** p < .001,extremely significant; ** p < .01,very significant, * 0.01≤p < .05,significant; ns indicates no significance.

Identification and gene structure analysis of BnCCR genes
Eight CCR genes were identified based on the ramie genome and designated as BnCCR1-8.BnCCR1, -2, -4, and -6 genes were anchored to chromosomes 5, 7, 1, and 10, respectively.Chromosome 13 contained BnCCR3 and BnCCR8 genes.Chromosome 2 contains BnCCR5 and BnCCR7 genes (Insert Table S5 here).Moreover, Analysis of the structure of BnCCR genes revealed significant differences in the number, length, and position of exons and introns (Figure 2a).BnCCR8 consists of two introns; BnCCR1 and BnCCR2 consist of four introns; BnCCR3, -4, -5, and -7 consist of five and BnCCR6 consist of eight introns, respectively.

Cloning and primary structure analysis of BnCCR genes
BnCCR1-8 genes were successfully cloned by PCR in this study (Figure S1).The CDS lengths of the eight BnCCR genes ranged from 879 to 1146 bp, encoded 292 to 381 amino acids.There were differences in protein molecular weight, isoelectric point, and subcellular location (Insert Table S5 here).The protein instability index of BnCCR proteins was between 28.63 and 39.51, indicating that all BnCCR genes encoded stable proteins.Unlike other BnCCR2 and BnCCR3 are located in the cytoplasm, unlike other BnCCR genes that are localized in the Golgi apparatus.Moreover, analysis of the transmembrane helices of BnCCR proteins revealed that BnCCR1 has one transmembrane helix, BnCCR6 contains two transmembrane helices, and none of the transmembrane helices in the other six BnCCR proteins (Figure S2).

Conservation and diversity of BnCCR proteins
The obtained BnCCR proteins contained the majority of the known primary structures of these enzymes, including the typical Rossmann fold and conserved Ser-Tyr-Lys catalytic triad (Table 1) (Chao et al. 2019).BnCCR2 and BnCCR3, Ser-His-Lys and Ser-Gln-Lys, respectively, instead of Ser-Tyr-Lys.The conserved motifs G-X-X-G-X-X-A and D-X-X-D have been reported to be involved in NAD(P) binding and adenine binding pocket stabilization (Chao et al. 2017a), with G-X-X-G-X-X-A having base mutations in BnCCR2 and BnCCR3 and D-X-X-D having base mutations in BnCCR6.In addition, the NADP-specificity motif R-(X) 5 -K is a key structure that distinguishes CCR from other NAD(H) SDRs (Pan et al. 2014).Among the eight BnCCR proteins, BnCCR2, -3, -5, and -8 do not contain this key motif.In addition, It is notable that the substrate binding motif (CCR-SBM) H-(X) 2 -K can be identified as universal signature sites for distinguishing CCRs from CCR-like proteins in land plants (Chao et al. 2019), and CCR-like proteins lose catalytic activity.H 2 0 2 was mutated to M/V/S/L in all BnCCR proteins, except BnCCR1.H-(X) 2 -K was not present in BnCCR2.R253 binds to a highly electronegative region of CoA (Chao et al. 2017b).All of the BnCCR proteins have substitutions at R253, in addition to BnCCR1 and BnCCR2.Table 1.Analysis of the conserved and active sites of BnCCR proteins.

Phylogenetic analysis of BnCCR proteins
To better understand the evolutionary relationships among BnCCRs and determine their classification, a phylogenetic tree was constructed for the reported CCR proteins from various plants (Figure 2b).BnCCR proteins were classified into three groups.Group I comprises genuine CCRs from terrestrial plants, BnCCR1 is rooted in this group.BnCCR2 and BnCCR3 clustered in Group II and were closely related to BpCCR1 (Zhang et al. 2015) and (Medicago sativa) MsCCR-like1 (Cui et al. 2022), forming a new CCR group in ramie, but with differences in the active sites of BnCCR2, BnCCR3, and BpCCR1.Group III included patients with BnCCR4, -5, -6, -7, and -8.Interestingly, BnCCR4 formed Group IIIa, BnCCR5 and BnCCR8 together with (Glycine max) GmCCR (So et al. 2010) formed a subgroup named Group IIIb.BnCCR6 and BnCCR7 formed Group IIIc.Moreover, Groups II, Group IIIa, and Group IIIc contained CCR-like proteins.

The expression patterns of BnCCR genes under abiotic stresses
To determine whether BnCCR genes are involved in abiotic stress responses based on their evolutionary relationships, the expression levels of eight BnCCR genes were analyzed using qRT-PCR.The BnCCR genes responded differently to stress with increasing Cd concentrations (Figure 4).The expression levels of BnCCR1 and BnCCR2 showed a trend of upregulation first and then downregulation; BnCCR1 at 20 mg/L Cd after treatment was 4-7 times higher than that at other Cd concentrations.The expression level of BnCCR3 showed a downregulation trend, followed by upregulation.The expression level of BnCCR4 was upregulated.The expression levels of BnCCR5 and BnCCR8 were first upregulated and then downregulated, and follow upregulated and then downregulated.BnCCR6 and BnCCR7 were first upregulated and then downregulated, followed by upregulation.BnCCR5 expression at 20 mg/L Cd after treatment was four times higher than that at other Cd concentrations.The BnCCR genes responded differently to stress with increasing NaCl concentrations (Figure 5).The expression level of BnCCR1 showed was initially upregulated and then downregulated.The expression levels of BnCCR2, -3, -5, -7, and -8 showed V patterns.The expression of BnCCR4 was downregulated, and BnCCR6 was upregulated, respectively.

BnCCR genes exhibit different expression levels in the phloem and xylem of the stem in ramie
The differential expression levels of eight BnCCR genes in the phloem and xylem of the stem during the seedling and vigorous growth periods were analyzed by qRT-PCR (Figure 6a).The expression levels of BnCCR2-8 in two tissues were significantly different during the two periods except for BnCCR1.At the seedling stage, the expression levels of BnCCR1, −2, 4-6 were significantly higher in the phloem than in the xylem, the expression levels of BnCCR7-8 were significantly lower in the phloem than in the xylem, and BnCCR3 was not significantly different between the two tissues.During the vigorous growth period, the expression levels of BnCCR1, −2, −4, −5, and -7 were significantly higher in the phloem than in the xylem, whereas the expression levels of BnCCR3, −6, and -8 were not significantly different between the two tissues.Notably, the expression of BnCCR2 in the phloem was 66 times higher than that in the xylem.

Correlation between the lignin content and the expression levels of BnCCR genes
Correlation analysis based on lignin content (Figure 6b) and the expression levels of eight BnCCR genes in the phloem and xylem of the stem during the seedling and vigorous growing periods (Table 2).In the phloem, the lignin content was not significantly different between the two periods.
In the xylem, the lignin content was significantly different between the two periods.The lignin content of the xylem was significantly higher than that of the phloem.During the seedling period, lignin content was significantly and positively correlated with the expression levels of BnCCR1, -5, -6, and -8 in the xylem, and lignin content was significantly and positively associated with the expression levels of BnCCR1, -4, -7, and -8 in the phloem, and was significantly and negatively associated with BnCCR3 and BnCCR5 in the phloem.During the vigorous growth period, lignin content was significantly and positively associated with the expression levels of BnCCR1, -2, and -5 in the xylem and phloem, and was significantly and negatively associated with BnCCR3, -4, -6, and -7 in the phloem.

Correlation between the flavonoid, lignin content and the expression levels of BnCCR2
Previous in vitro biochemical studies have shown that BnCCR2 can catalyze cinnamoyl-CoA and sinapoyl-CoA, which may not only participate in lignin biosynthesis (Tang et al. 2022).Therefore, the correlation analysis was based on the flavonoid content, lignin content, and expression levels of BnCCR2 in ramie during the seedling, vigorous growth, and maturation periods.The highest expression of BnCCR2 during the vigorous growing period was 14 times higher than that during the seedling period (Figure 7a).The flavonoid (Figure 7b) and lignin contents (Figure 7c) gradually increased with the growth of ramie.The expression levels of BnCCR2 in the three periods were positively correlated with both lignin and flavonoid content, whereas lignin content and flavonoid content were significantly negatively correlated (Figure 7d), indicating that BnCCR2 is closely related to lignin and flavonoid biosynthesis, and there is a significant competitive relationship between lignin and flavonoids.

Evolutionary characteristics of the BnCCR homologous genes
Diverse CCR families have been reported in some plant species.There are 11 CCRs in Populus tomentosa (Chao et al. 2017b), seven in Arabidopsis thaliana (Raes et al. 2003), 33 in Oryza sativa (Park et al. 2017), 10 in Eucalyptus grandis (Carocha et al. 2015), 30 in Medicago sativa (Cui et al. 2022) and nine in Populus trichocarpa (Shi et al. 2010).Eight BnCCR genes were identified and cloned into ramie plants.There are significant differences in the sequence and structure, which can be divided into three groups.Group I consists of genuine CCRs isolated from terrestrial plants.These proteins are thought to be involved in lignin biosynthesis, as PtoCCR1 and PtoCCR7 (Chao et al. 2017b), AtCCR1  and AtCCR2 (Lauvergeat et al. 2001), and OsCCR1 (Park et al. 2017) were included in this group, all of which are involved in lignin biosynthesis according to experimental evidence.BnCCR1 was rooted in this group along with all previously reported genuine CCRs in which the NAD(P)-binding and NADPspecificity motifs, as well as the CCR signature motif, were fully conserved.BnCCR1 has been confirmed to belong to bona fide CCR family and is involved in lignin biosynthesis through biochemical functions in vitro (Tang et al. 2022).
BnCCR2 and BnCCR3 clustered in Group II and contained BpCCR1.BpCCR1 overexpression increased lignin content by up to 14.6%, and downregulation of its expression decreased lignin content by 6.3%.Modification of BpCCR1 expression leads to conspicuous changes in wood characteristics, including xylem vessel number, arrangement, and secondary wall thickness.The height of the transgenic trees was also significantly influenced by the modification of BpCCR1 genes (Zhang et al. 2015).Therefore, we speculate that BnCCR2 and BnCCR3 play different roles in the xylem and phloem of ramie.The obtained BnCCR proteins contained the majority of the known primary structures of these enzymes, including G-X-X-G-X-X-A, the NADP specificity motif R-(X) 5 -K and conserved Ser-Tyr-Lys catalytic triad (Pan et al. 2014), BnCCR2 and BnCCR3 did not contain or had substitution at this key motif.Notably, the substrate binding motif (CCR-SBM) H-(X) 2 -K is only detected in genuine CCRs, which can be identified as universal signature sites for distinguishing CCRs from CCR-like proteins in land plants (Chao et al. 2019), CCR-like proteins lose catalytic activity.Mutants H208M, H208V, and H208Y of PtoCCR7 had no detectable activity toward either feruloyl-CoA or sinapoyl-CoA (Chao et al. 2017b).There is no H-(X) 2 -K in BnCCR2, but BnCCR2 was characterized by sinapoyl-CoA and cinnamoyl-CoA reductase activity, mutants H202S of BnCCR3 without any detectable catalytic activity in hydroxycinnamoyl-CoA esters (Tang et al. 2022).In addition, the subcellular location and template used for constructing the threedimensional models of BnCCR2 and BnCCR3 proteins were different from those of other BnCCR proteins.Therefore, we considered BnCCR2 may not be involved in lignin synthesis alone.Group III contains GmCCR and CCR-like proteins.GmCCR is involved in resistance mechanisms during abiotic stresses in plants (So et al. 2010), and SmCCR-like (Chao et al. 2017b), (Panicum virgatum) PvCCR-like (Escamilla-Trevino et al. 2010) and PtoCCR2, -3, -5, and -6 proteins (Chao et al. 2017b) have been identified in this cluster, which do not have CCR activity.BnCCR4, -5, -6, -7 and -8 belonged to this cluster.Whether these genes participate in ramie stress resistance or in other functions of these proteins requires further research.

The different expression patterns of BnCCR genes under abiotic stresses
Soil pollution caused by cadmium (Cd) is a major concern.Phytoremediation is a popular technology used for the remediation of Cd-contaminated soils.Ramie is a preferred crop in the implementation plan of the industrial Structure Adjustment Project with Serious Heavy Metal Pollution in Hunan, China (Chen, Zhu, and Xiong 2020).Phenylpropanoid biosynthesis has been identified as a critical pathway in response to Cd stress in Salix matsudana.In this pathway, the Cd-induced gene cinnamoyl-CoA reductase 1 (SmCCR1) from Salix matsudana increases lignin content and enhances Cd tolerance in transgenic poplar calli (Yu et al. 2023).qRT-PCR showed that abiotic stress could lead to different responses in BnCCR genes, but the expression of only BnCCR4 was upregulated with increasing Cd concentration.Thus, we suggest that BnCCR4 is involved in cadmium stress in ramie plants.
Several studies have shown that CCR participates in coping with environmental stresses in plants.The early response genes to salt stress in the roots of melon seedlings include CCR and the transcript factor MYB (Wei et al. 2013).CCR11 is upregulated under salt-deficient conditions in Populus trichocarpa (Hori et al. 2020).The expression of BnCCR4 was downregulated, and BnCCR6 was upregulated.Considering the expression levels of CCR genes under Cd and salt stress, we speculate that BnCCR4 is involved in the abiotic stress response in ramie.These results lay the foundation for further clarification of the molecular mechanisms underlying abiotic tolerance in ramie.

The different correlation between the lignin content and the expression levels of BnCCR genes in the xylem and phloem of the stem in ramie
In Populus trichocarpa, Shi et al. identified 11 CCRs, and PtrCCR2 showed high expression in differentiating xylem and significantly less in other tissues (Shi et al. 2010).RNAi suppression of PtrCCR2, the only CCR member highly expressed in the stem-differentiating xylem, causes a reciprocal reduction in stem-differentiating xylem protein CAD activity (Yan et al. 2019).Therefore, it is necessary to study the regulatory mechanisms of BnCCR genes in the xylem and phloem of ramie.The lignin content was significantly and positively correlated with the expression levels of BnCCR1 in the phloem and xylem of ramie during the two growth periods.These results further suggested that BnCCR1 is involved in lignin synthesis in ramie.In addition, the expression level of BnCCR5 was significantly correlated with lignin content during the two periods of ramie growth.Thus, we suggest that BnCCR5 is also involved in the synthesis of ramie lignin.

The different correlation between the flavonoids content, lignin content and the expression levels of BnCCR2 genes in ramie
The BnCCR2 gene was highly expressed in the phloem during vigorous growth.The expression level of BnCCR2 positively correlated with lignin and flavonoid content at all three time points.Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways (Dardick et al. 2010).The expression level of CCR was significantly higher than that at 36 h at the 120 h germination stage, promoting phenolic accumulation (Chu et al. 2020).We suggest that BnCCR2 may be closely related to lignin and flavonoid synthesis, and that BnCCR2 plays different roles in the xylem and phloem of ramie during different periods.

Application prospect of BnCCRs in ramie breeding
CCR has been used to study the regulation of lignin and flavonoid content, cell wall generation, and antibacterial and stress resistance.Studies have shown that transgenic rice plants overexpressing OsCCR10 show improved drought tolerance at the vegetative stages of growth, as well as higher photosynthetic efficiency, lower water loss rates, and higher lignin content in roots compared to non-transgenic controls.In contrast, CRISPR/Cas9-mediated OsCCR10 knockout mutants exhibit reduced lignin accumulation in the roots and reduced drought tolerance (Bang et al. 2022).Four BnCCR genes were screened and identified based on their sequence, structure, evolution, response to stress, tissue expression, and correlation with the lignin and flavonoid contents of ramie.BnCCR1 and BnCCR5 are related in lignin synthesis, and BnCCR4 is related to abiotic stress, respectively.Previous studies have shown that BnCCR1 belongs to bona CCR, which catalyzes cinnamoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA, confirming that BnCCR1 is indeed involved in lignin biosynthesis.This lays the foundation for verifying the function of this gene in ramies.BnCCR2 is also associated with the synthesis of flavonoids and lignin, which may play different roles in the xylem and phloem of ramie at different stages, thus providing a new reference for studying the evolution of the CCR protein family.Therefore, it will be fascinating to check the function of CCR-likes and to compare the structural and functional differences between CCR and CCR-like proteins in future studies.Furthermore, the overall quality should be considered when a genetic approach is implemented to target ligninreduced ramie.

Conclusions
In this study, eight BnCCR genes were screened and identified based on sequence characteristics, gene structure, phylogenetic relationships, stress response to abiotic treatment, and correlation of lignin and flavonoid content with the expression levels of genes in the phloem and xylem of ramie stems.BnCCR1 and BnCCR5 are related to lignin synthesis, BnCCR4 is related to abiotic stress, and BnCCR2 is closely related to both lignin and flavonoid synthesis, and may play different roles in the xylem and phloem of ramie at different periods.The functions of the other genes require further investigation.This not only provides a new reference for studying the evolution of the CCR protein family, but also provides a basis for the breeding of multifunctional varieties of ramie.

Figure 2 .
Figure 2. Gene structure of BnCCR, CDS, UTR, and introns are represented by black rectangle, gray rectangles, and gray lines, respectively (a).Phylogenetic relationship of the CCR proteins of ramie and other plants (b).

Figure 3 .
Figure3.Structural modeling analysis of BnCCR proteins.The extended strand is indicated by a yellow ribbon, the alpha helix is indicated by a pink ribbon, the beta-turn and the random coil is indicated by a white ribbon, the R-(X) 5 -K motif is indicated by red stick amino acid residues, the conserved sites S-Y-K of the short chain dehydrogenase superfamily is indicated by blue, earthy yellow, and green stick amino acid residues, respectively.The important substrate binding site H202 of CCR is indicated by a green round ball, and NAP in BnCCR1 is shown in light blue rod amino acid residues.

Figure 4 .
Figure 4.The expression profile of BnCCR genes in stem under cd stress.

Figure 5 .
Figure 5.The expression profile of BnCCR genes in stem under NaCl stress.

Figure 6 .
Figure 6.The expression profile of BnCCR genes in the xylem and phloem of the stem at the two periods (a), the lignin content profile of the xylem and phloem of the stem at the two periods (b).

Figure 7 .
Figure 7.The expression profile of BnCCR2 in ramie during three periods (a), histogram of the flavonoid content in ramie during three periods (b), histogram of the lignin content in ramie during three periods (c), correlation coefficients of the lignin content, flavonoid content and the expression level of BnCCR2 (d).S: seedling period, V: vigorous growing period, M: mature period.

Table 2 .
Correlation coefficients of lignin content and the expression levels of BnCCR genes.