The NAC transcription factor LuNAC61 negatively regulates fiber development in flax (Linum usitatissimum L.)

Dongwei Xie ,Jing Li ,Wan Li ,Lijun Sun ,Zhigang Dai ,Wenzhi Zhou ,Jianguang Su,Jian Sun#

1 School of Life Sciences, Nantong University, Nantong 226019, China

2 Institute of Crop Cultivation and Tillage, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

3 Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410221, China

4 Jiangsu Sanshu Biotechnology Co. Ltd., Nantong 226001, China

Abstract Flax is a crucial fiber crop that exhibits excellent textile properties and serves as a model plant for investigating phloem fiber development. The regulation of multiple genes significantly influences fiber development,notably involving NAC (NAM,ATAF1/2,CUC2) transcription factors in forming the fiber secondary cell wall (SCW).Overexpression of LuNAC61 in flax resulted in sparse top meristematic zone leaves and significantly reduced stem cellulose content. Scanning electron microscopy and staining observations revealed a significant reduction in fiber bundles. β-Glucuronidase (GUS) staining analysis demonstrated high activity of the LuNAC61 promoter in the bast fibers of the flax stem. Additionally,several members of the LuPLATZ and LuCesA families exhibited significant coexpression with LuNAC61. Subcellular localization indicated the presence of LuPLATZ24 protein in the nucleus and cytoplasm,LuNAC61 protein exclusively in the nucleus,and LuCesA10 in the nucleus and endoplasmic reticulum. LuPLATZ24 positively regulates LuNAC61,whereas LuNAC61 negatively affects LuCesA10,suggesting the involvement of a metabolic network in regulating flax fiber development. In conclusion,this study provides a critical opportunity for a comprehensive and in-depth analysis of the mechanisms governing flax fiber development and the potential use of biotechnology to enhance flax fiber yield.

Keywords: flax,fiber development,LuNAC61,gene function,gene interaction

1.Introduction

The NAC (NAM,ATAF1/2,CUC2) family of plant-specific transcription factors (TFs) has been extensively studied,and its members have been identified in various plant species. In upland cotton,142GhNACgenes exhibit distinct expression patterns across diverse stages of fiber growth and development (Sunet al.2018). Notably,three of these genes share high homology withArabidopsis NST1,and potentially participate in regulating secondary wall formation (Zhonget al.2007).GhFSNl,a NAC transcription factor gene,can form homodimers and activate itself by binding to downstream gene promoters.Its direct target genes,includingGhIRX12,GhGUT1,GhMYBl1,GhKNL1,andGhDUF231L1,are closely associated with fiber secondary walls. In the context of cotton fiber secondary wall formation,GhFSN1actively regulates the strength,length,and quality traits of cotton fiber by activating the downstream genes relevant to these secondary walls (Zhang Jet al.2018). Sunet al.(2018)proposed that theGhFSN5gene acts as a negative regulatory transcription factor for plant secondary cell walls (SCW) formation. Through heterologous expression inArabidopsis,transgenic lines exhibited lower lignin and cellulose contents in the roots and stems compared to the wild type (WT). Consequently,the genes involved in synthesizing lignin,xylan,and cellulose,as well as certain SCW-related transcription factors (TFs),experienced significant downregulation.

Flax is an essential fiber crop that boasts fibers with superior textile properties (Mokshinaet al.2020;Rituet al.2022). These fibers exhibit moisture absorption,bacteriostatic and antistatic functions,and exceptional flame-retardant effects. They are used in producing various items like clothing,decorative fabrics,bedding,and automotive supplies,and are experiencing an increasing market demand. As a small,diploid,selfpollinating plant with a small genome,flax serves as a model plant for studying phloem fiber development.Understanding the molecular mechanisms governing fiber growth and development is crucial for enhancing flax fiber quality and yield. During the rapid growth stage of flax,a distinctive boundary point emerges. The cells above this point cease elongation,while those below it undergo cell wall thickening. As the plant matures,these boundary points gradually ascend along the stem,ultimately vanishing upon the completion of stem development during the flowering stage (Hobson and Deyholos 2013).The thickening of SCW involves significant cellulose deposition. Therefore,the rapid growth and flowering stages are pivotal in determining flax fiber quality and yield.

The complex development of flax bast fiber SCW is influenced by numerous gene expression regulators includingCesA,Susy,BGALs,UGT,and others (Roachet al.2011;Hobson and Deyholos 2013;Khotylevaet al.2014;Mokshinaet al.2014;Xieet al.2022). However,our knowledge of the molecular biological foundation for flax fiber development is lagging. Advancements in research techniques,including the completion of the flax genome sequencing project and improvements in bioinformatics,biochemistry,molecular biology,and genetics,are continually improving our understanding of flax fiber development at the molecular level. While progress has been made in uncovering key regulatory genes for flax fiber development,few reports exist on the influences of transcription factors in this process. Many studies inArabidopsisand cotton have demonstrated the role of NAC in regulating fiber secondary walls,thereby affecting fiber yield and quality. However,flax is a significant fiber industrial crop that lacks substantial research on NAC transcription factors,and there have been no reports regarding NAC family members in flax associated with fiber development and secondary wall formation. Secondary wall development significantly influences the quality and yield of flax fiber. Hence,the identification and validation of NAC family members related to flax fiber development and secondary wall formation are imperative.

An examination of the flax genome database revealed 184 NAC family members,which were subjected to phylogenetic analysis. Our previous research revealed that functional deficiency ofLuUGT175in flax led to slow growth,substantial stem lignification,and abnormal fiber lignification.LuNAC61was identified as the upstream negative regulatory factor ofLuUGT175. Bothinvitroandinvivovalidation confirmed their direct interaction(Xieet al.2022),soLuNAC61was selected for further investigation. Its role in regulating flax fiber SCW development to influence overall flax fiber development was verified. Notably,LuPLATZsandLuCesAsare closely associated with plant fiber development (Khotylevaet al.2014;Mokshinaet al.2014;Handeet al.2017).To explore the fiber development regulatory network,gene co-expression analysis and interaction verification were conducted between theLuNAC61,LuPLATZs,andLuCesAsfamily members. This preliminary exploration paved the way for the creation of transgenic materials,aiming to enhance flax fiber quality and yield by establishing a theoretical framework and the necessary genetic resources.

2.Materials and methods

2.1.Database search for NAC members in flax

A total of 138ArabidopsisNAC protein sequences were downloaded from theArabidopsisGenome Website(https://www.arabidopsis.org/). The HMM file (PF02365,NAM.HMM) of the NAC gene family conserved domain hidden Markov model was downloaded from Pfam (http://pfam.xfam.org/). The flax genome protein sequence was downloaded from the Figshare website (https://figshare.com/articles/dataset/Annotation_files_for_Longya-10_genome/13614311). Using TBtools (https://www.tbtools.com/) within the “Simple HMM Search” module,the HMM file and flax genomic protein sequence were searched for flax genomic NAC protein data. The resulting file was validatedviathe CDD,Pfam,and SMART databases to confirm the conserved domains,with manual removal of the incomplete PSPG box sequence. This process generated the flax NAC family member protein sequences.

2.2.Phylogenetic analysis of NAC family members in flax

The 184 flax NAC protein sequences and 138ArabidopsisNAC protein sequences were aligned using ClustalX 1.83 software. A phylogenetic tree was constructed using MEGA 4.1 software with the NAC protein sequence files from the ClustalX comparison,employing the neighborjoining method with a bootstrap value of 1,000.

2.3.Analysis of LuNACs expression patterns during the development of flax fibers

For transcriptome analysis,the WT flax variety YM6 was cultivated in the phytotron of Nantong University,simulating natural growth conditions (24°C/18°C,16 h light/8 h dark,light intensity 6,000 lux,humidity 50%).Stems were harvested at the fir-like stage (4 weeks after seedling emergence),rapid growth stage (6 weeks after seedling emergence),and flowering stage (8 weeks after seedling emergence). Each stage comprised three biological replicates,and subsequent transcriptome analyses were conducted following the methodologies detailed in our previous publication (Xieet al.2019).

2.4.Construction of the LuNAC61 overexpression vector and β-galactosidase (GUS) fusion vector

TheLuNAC61gene spans 1,586 bp,comprising three exons and two introns,and encoding 411 amino acids.The target gene fragment was acquiredviadouble enzyme digestion of the pBSK-NACs cloning vector (Jiangsu Sanshu Biotechnology Co.,Ltd.,China) followed by electrophoretic recovery. Similarly,the pCAMBIA1301 vector was digested with the same enzyme to obtain and recover the fragment containing the 35S promoter. The T4 ligase was used to link the target gene fragment to the pCAMBIA1301 vector fragment,transforming the linking system into competentE.coliDH5awhich were plated on LB solid medium. Positive clones were verified through monoclonal selection,growth in LB liquid medium,plasmid extraction,PCR,and enzymatic digestion. The recombinant eukaryotic expression vector PCAMBIA1301-NACs was introduced intoAgrobacteriumtumefaciensGV3101,using bacterial shaking cultures from selected positive colonies for later use. Primer design relied on the promoter reference sequence from the flax genome database. Promoter fragment cloning and sequencing employed flax DNA as the template,with the correctly sequenced promoter sequence inserted upstream of theGUSgene in the pBI101 vector to construct the GUS vector used as a promoter-less control. The primer sequences for promoter amplification were:LuNAC61(forward: 5′-AGGAAACAGAAAAGCAAT-3′,and reverse:5′-TGAGGGTAGTAGCAGAAG-3′). Histochemical GUS determination was performed as described earlier with slight modifications (Jeffersonet al.1987).

2.5.Transformation of LuNAC61 and promoter::GUS(empty vector as control) in flax

Tissue culture and genetic transformation methods were employed according to our previous study (Xieet al.2022).Using the hygromycin gene (HPT) as the target,specific primers (HPT-F: 5′-GTTAGCGTCAGCGAGGATG-3′;HPT-R: 5′-AGGAGATCGGCAAGGCCACC-3′;PCR product size: 610 bp) were designed to confirm the transformed positive plants.

2.6.Scanning electron microscopy (SEM) and staining observations

SEM and staining observations utilized the WT flax variety YM6 andLuNAC61overexpression plants (OE4 and OE6). These materials were cultivated at the phytotron of Nantong University,mimicking natural growth conditions(24°C/18°C,16 h light/8 h dark,light intensity 6,000 lux,humidity 50%). Stem samples (10 cm above the flax cotyledonary node) were obtained during the rapid growth and flowering stages. These stems underwent M?ule and Wiesner staining for subsequent observations,following the procedures detailed in our prior publication (Xieet al.2022).

2.7.GUS staining analysis

A total of 2-mL sterile centrifuge tubes containing 1 mL of GUS dye solution were prepared in a sterile environment.The roots,stems,and leaves of both promoter::GUS and empty vector-positive seedlings (grown at 24°C/18°C,under 16 h light/8 h dark conditions,with a light intensity of 6,000 lux and 50% humidity) were dissected using sterile tweezers and scalpels. These plant materials were submerged in the GUS dye solution within the tubes. To prevent cross-contamination,each sample was sterilized with a high-temperature alcohol lamp after dissection and then incubated at 37°C on a shaking table set to 150 r min–1overnight. Subsequently,the GUS dye solution was replaced with 1 mL of 70% absolute alcohol for decolorization. The samples were placed on a shaking table at room temperature,set to 100 r min–1,and fresh 70% anhydrous alcohol was added hourly until the green color of the flax tissue was completely removed,leaving a generally white background. Finally,the flax tissues were removed,and photographs were taken.

2.8. Determination of cellulose content

The WT flax variety YM6 andLuNAC61overexpression plants (OE4 and OE6) were used for cellulose content analysis. The stem samples were collected (obtained from 10 cm stems above the flax cotyledonary node) during the rapid growth and flowering stages (24°C/18°C,16 h light/8 h dark,light intensity 6,000 lux,humidity 50%).Each sample of approximately 1.5–2 g underwent drying at 80°C until a constant weight was achieved,and the sample was then transferred to a dryer to cool to room temperature and ground into powder. About 0.1 g of this powder was weighed precisely and treated with a solution containing 75 g of 0.5 mol L–1NaOH solution,boiled for 4 h to remove hemicellulose,and filtered. The resulting residue from the filter was dried. A 15 mL sample was subjected to a 75% sulfuric acid hydrolysis process and stirred continuously to prevent lumps,followed by a 10-h settling period. Post-settling,400 mL of deionized water was added,and the sample was then boiled for 4 h,and filtered. The container was thoroughly washed to remove acid,with all filtrates collected and adjusted to reach a final volume of 1,000 mL. A total of 50 mL of filtrate were transferred to an iodine measuring flask. Methyl orange indicator drops were added,and the solution was sequentially neutralized with 1 mol L–1NaOH solution to achieve alkaline conditions and then returned to acidic conditions by adding 1 mol L–1HCl solution. Subsequently,50 mL of 1 mol L–1NaOH solution was combined with 20 mL of 0.1 mol L–1iodine standard solution,left to stand for 2 h,and weakly acidified by adding 1 mol L–1HCl solution,then titrated using sodium thiosulfate with starch as the indicator. This process was accompanied by a blank run to record the volume of consumed sodium thiosulfate. Three biological replicates were processed in parallel for each sample period.

2.9.Quantitative real-time PCR

Samples were collected during two critical stages,the rapid growth and flowering stages,that are pivotal for flax fiber development (Yuanet al.2019;Yuet al.2021).The internal control used for qRT-PCR was 18S (NCBI Accession: EU307117),and the specific target genes and 18S primers are shown in Appendix A. Relative expression levels were determined using the 2–??CTmethod and calculated as ΔΔCT=(CT,target–CT,internalcontrol)timex–(CT,target–CT,internalcontrol)time0(Livak and Schmittgen 2001). All qRT-PCR experiments were conducted in triplicate.

2.10.Subcellular localization analysis

The complete CDS sequences ofLuPLATZ24,LuNAC61,andLuCesA10were cloned from flax into pRI101 vectors,so they were fused with the green fluorescent protein(GFP) gene under the control of the CaMV 35S promoter,transforming into the plasmids pRI101-LuPLATZ24-GFP,pRI101-LuNAC61-GFP,and pRI101-LuCesA10-GFP,respectively. These vectors were then introduced into GV3101,following our detailed methodologies as described previously (Xieet al.2022,2023).

2.11.Dual-luciferase reporter gene experiment(Dual-Luc)

The 62SK-LuPLATZ24-pGreenII0800-LUC-LuNAC61(pro)and 62SK-LuNAC61-pGreenII0800-LUC-LuCesA(pro)recombinant vector was the detection group. 62SKpGreenII0800-LUC-LuNAC61(pro) and 62SKpGreenII0800-LUC-LuCesA(pro) were the control group.If the promoter ofLuNAC61interacted withLuPLATZ24,and the promoter ofLuCesAinteracted withLuNAC61,the N-terminal (N-Luc) and C-terminal (C-Luc) of the fluorescein enzyme were brought close to each other in space and assembled into a complete fluorescein enzyme that was active in decomposing the substrate and producing fluorescence,following our detailed methodologies as described previously (Xieet al.2022).

3.Results

3.1.NAC family members in the phylogenetic analysis

The 322 NAC family members,comprising 184LuNACsand 138AtNACs,were categorized into 15 established and conserved phylogenetic subfamilies (NAC1,NAC2,TERN,ONNAC022,ANAC011,OsNAC7,NAM,OsNAC8,TIP,ANAC001,ATAF,AtNAC3,NAP,ANAC063,and ONAC003),along with four unclassified subfamilies.Evolutionary analysis revealed 19 groups of flax NAC members. The largest group comprised 50 members(27.17%),categorized as unclassified. Groups with significant membership included ONAC022,OsNAC7,and NAP,each including 17 members. The remaining groups,namely NAC1,NAC2,TERN,ANAC011,NAM,OsNAC8,TIP,ANAC001,ATAF,AtNAC3,and ONAC003,contained 6,13,3,8,13,4,6,7,9,and 2LuNACs,respectively.Notably,the ANAC063 group did not include any flax NAC members (Fig.1).

3.2.LuNAC61 gene functional analysis

Phylogenetic analysis showed that 17LuNACs andArabidopsisANAC012were categorized under the OsNAC7 subgroup,withLuNAC13,LuNAC61,LuNAC112,LuNAC125,andANAC012placed within the same branch. Initial research findings revealed that significant overexpression ofANAC012inArabidopsis thalianainhibited lignocellulose secondary wall deposition in stems,increasing the cell wall thickness in xylem vessels. Simultaneously,it led to reduced cellulose composition in the stems and roots,likely due to a cellulose formation defect.ANAC012has been established as a negative regulator of cellulose secondary wall thickening (Koet al.2007). Therefore,we speculated thatLuNAC13,LuNAC61,LuNAC112,andLuNAC125might share similar functions withANAC012.We observed varying expression trends ofLuNAC13,LuNAC61,LuNAC112,andLuNAC125at different stages of flax stem development. Among them,LuNAC13andLuNAC112maintained consistent expression levels during the fir-like stage (4 weeks),rapid growth stage(6 weeks),and flowering stage (8 weeks).LuNAC112initially increased and then decreased during flax stem development.LuNAC61exhibited its highest expression during the fir-like stage,significantly declining during the rapid growth and flowering stages. Notably,following the law of secondary wall thickening in flax phloem fiber cells,the period from the rapid growth stage to the flowering stage represents a crucial phase for secondary wall thickening and cellulose accumulation in flax phloem fibers. Therefore,theLuNAC61expression significantly declined during these two critical periods,aligning with a potential negative regulatory effect on fiber development.Subsequently,we screened this gene for further validation(Appendix B). To confirm the function of theLuNAC61gene,we created aLuNAC61overexpression vector,conducted flax genetic transformation,assessed the cellulose content in the stems of the positive materials,and carried out SEM and staining observations. To identify flax materials that overexpress theLuNAC61gene (Appendix C),PCR identification revealed 10 positive lines from the genetic transformation (Appendix C). Two of these lines (OE4 and OE6) were selected for mass propagation and subsequent experimental analysis.During the rapid growth stage of flax,the overexpression plants exhibited reduced thickness compared to the WT plants. Young leaves in the top meristem were sparse,resulting in an overall frail appearance (Appendix C).

This study focused on examining fiber changes in the stems of flax plants with overexpression. We conducted SEM and comparative dyeing observations on the stem cross-sections of overexpression lines (OE4 and OE6) and the WT. The rapid growth phase is crucial for determining flax fiber quality and the initial cellulose accumulation in stems. Therefore,we made observations of the stem cross sections of WT and overexpression plants during rapid growth with a scanning electron microscope. The WT flax stems began accumulating cellulose and forming fiber bundles (Fig.2-A–C),but there was little accumulation of cellulose in the stems of OE4 and OE6 overexpression plants,and only a small number of fiber bundles were formed,as shown by the red arrow in Fig.2-D–I. Notably,WT flax accumulated a significant amount of cellulose and formed tightly connected fiber bundles during rapid growth,while OE4 and OE6 overexpression plants showed reduced cellulose accumulation and a smaller number of fiber bundles(Appendix D).

Upon entering the flowering stage,flax undergoes a transition from vegetative growth to the formation of cellulose and bundle fibers in the stem,a feature that remains relatively constant thereafter. To assess this,Wiesner and M?ule staining were conducted on flax stems during the flowering stage (Fig.3). M?ule staining complements Wiesner staining by coloring lignin,aiding in the observation of fiber quantity within flax stems due to the presence of some lignin in the fibers. This examination revealed densely arranged fiber bundles in WT flax stems (Fig.3-A and B). In contrast,the overexpression plants (OE4 and OE6) displayed significantly fewer fiber bundles that exhibited a looser arrangement (Fig.3-C–F). This observation indicates a significant reduction in flax fiber content in overexpression plants compared to WT plants. This deduction aligns with the cellulose content determinations in both WT stems and overexpressing plants (Appendix D).

Fig. 2 Scanning electron microscopy observations of a flax stem cross-section during the rapid growth stage (6 weeks).A–C,wild type (WT). D–F,overexpression of plant line 4 (OE4).G–I,overexpression of plant line 6 (OE6). F,fiber. X,xylem.

Fig. 3 Observations of cross-sectional staining of flax stems at the flowering stage (8 weeks) using compound microscopy and electron microscopy. A and B,wild type (WT). C and D,overexpression of plant line 4 (OE4). E and F,overexpression of plant line 6 (OE6). A,C,and E,Wiesner staining. B,D,and F,M?ule staining. F,fiber. X,xylem.

3.3.Analysis of LuNAC61 promoter activity in flax

The activity of theLuNAC61promoter was investigated by fusing a 2 kb upstream region to the GUS reporter gene,which was then transformed into flax. An analysis of the stems,leaves,and roots of positive plants (8 weeks) revealed that theLuNAC61promoter was inactive in roots (Fig.4-A) and exhibited relatively low activity in the stem cortex (Fig.4-B). Notably,theLuNAC61promoter showed high activity specifically in the bast fibers of the flax stem. GUS staining of transverse and longitudinal sections of the stem(8 weeks) displayed a significant bluish tint in the bast fibers compared to the empty vector control,confirming its heightened activity in these fibers (Fig.4-C and D).Furthermore,the activity of the promoter was greater in new leaves than in old leaves (Fig.4-E and F). No GUS activity was evident in any part of the control plant using the empty vector control (Fig.4 G–L).

3.4.Screening and identification of genes interacting with LuNAC61

NAC family members regulate downstream gene expression to impact plant development and growth,and they also cooperate with other transcription factors.In the transcriptome of cotton bolls at 10 days after anthesis,some NAC and PLATZ family members displayed downregulated expression. These factors primarily participate in plant secondary metabolism and fiber development (Handeet al.2017). To understand howLuNAC61regulates flax fiber development,we screened 28 LuPLATZ family members from the flax genome (Appendix E),and analyzed the coexpression network of 184LuNACgenes and 28LuPLATZgenes in the transcriptome. This analysis revealed thatLuNAC61forms a direct coexpression regulatory network with fiveLuPLATZfamily members(LuPLATZ1,LuPLATZ4,LuPLATZ10,LuPLATZ16,andLuPLATZ24). The coexpression coefficient betweenLuPLATZ24andLuNAC61exceeded 0.8,indicating a significant coexpression relationship (Fig.5-A;Appendix F). Furthermore,withLuNAC61overexpression in flax,the number of fiber bundles in transgenic flax stems decreased significantly. This suggests a regulatory relationship betweenLuNAC61and cellulose synthase.To confirm this relationship,we screened 41 CesA family members from the flax genome (Appendix G). An analysis of the coexpression gene network of 184LuNACand 41LuCesAgenes in the transcriptome revealed thatLuNAC61forms a direct coexpression regulatory network with 22LuCesAfamily members. Among them,six members of theLuCesAfamily (LuCesA6,LuCesA10,LuCesA20,LuCesA29,LuCesA35,andLuCesA40)had co-expression coefficients withLuNAC61>0.8,indicating significant coexpression relationships (Fig.5-B;Appendix H). We conducted qRT-PCR analysis of theLuPLATZandLuCesAfamily members that were significantly coexpressed (coexpression coefficients>0.8)withLuNAC61in the stems during the rapid growth and flowering periods of the WT and overexpression lines.The results suggested thatLuNAC61andLuPLATZ(LuPLATZ24) have positive regulatory relationships,whileLuNAC61and severalLuCesAgenes (LuCesA6,LuCesA10,LuCesA20,LuCesA29,LuCesA35,andLuCesA40) have negative regulatory relationships(Appendix C).

Fig. 4 LuNAC61 expression was detected by staining for GUS reporter gene activity in 8-week-old flax plants expressing the promoter (LuNAC61)::GUS (A–F) and the empty vector control(G–L) constructs. A and G,root. B and H,stem. C and I,longitudinal section of the stem. D and J,cross-section of the stem. E and K,young leaf. F and L,old leaf.

Based on the qRT-PCR results,LuPLATZ24,LuNAC61,andLuCesA10were selected for subcellular localization and interaction analysis. Using the fusion reporter gene method,GFP fused with the target proteins was transiently expressed inNicotianabenthamianaleavesvia Agrobacteriumtumefaciensinfection for the subcellular localization experiments. Laser confocal microscopy revealed the localization of the GFP-fused proteins,with LuPLATZ24 in the nucleus and cytoplasm,LuNAC61 exclusively in the nucleus,and LuCesA10 in the nucleus and endoplasmic reticulum. Notably,all proteins were found in the nucleus (as shown by the arrow in Fig.6-A).To confirm the interactions amongLuPLATZ24,LuNAC61,andLuCesA10,Dual-Luc assays were employed. The fluorescence signal of the tobacco tissue co-transfected withLuPLATZ24andLuNAC61(Pro) was significantly stronger than that of the empty vector and LuNAC61(Pro),indicating that the transcription factorLuPLATZ24has a promoting effect on theLuNAC61promoter. In contrast,co-transfection ofLuNAC61andLuCesA10(Pro) showed significantly weaker fluorescence than the co-transfection of an empty vector andLuCesA10(Pro),indicating a notable inhibitory effect ofLuNAC61on theLuCesA10promoter (Fig.6-B). However,there was no direct interaction observed betweenLuPLATZ24andLuCesA10(Pro). These results suggest a regulatory network whereLuPLATZ24upregulatesLuNAC61expression,subsequently leading toLuNAC61inhibitingLuCesA10expression,and ultimately influencing flax fiber development.

4.Discussion

4.1.NAC genes regulate the development of plant fiber SCW

The NAC gene functions have been studied in numerous crops. The different NAC family members play different roles in plant secondary wall formation,with some members promoting its formation,while others inhibit it. For example,the NAC transcription factorSND1inArabidopsisis a key transcription switch regulating secondary wall formation. SND1 is specifically expressed in the interfascicular fibers ofArabidopsisstems.Inhibiting its expression results in thinner secondary walls,while its overexpression leads to substantial wall deposition in fibers (Zhonget al.2006). Conversely,ANAC012overexpression significantly hampers fiber secondary wall deposition inArabidopsis. It increases xylem vessel cell wall thickness but reduces the cellulose contents in stems and roots.Tuttleet al.(2015) demonstrated the presence of ten genes closely resembling the transcription regulatorsNST1,SND2,andSND3fromArabidopsisSCW in cotton fiber expression. Similarly,MacMillanet al.(2017) compared the cotton xylem,pith,and developing ovule fiber transcriptomes,and identified several NAC transcription factors homologous toArabidopsis NST1/2/3,indicating their regulatory roles in cotton fiber secondary wall formation.Flax is a model crop for fiber development studies,although previous reports on NAC transcription factors regulating fiber secondary wall formation are lacking. This study marks the initial functional verification of flaxLuNAC61,which is highly akin toArabidopsisANAC012. OverexpressingLuNAC61in flax significantly reduced the fiber bundles in stems compared to the WT,affirming the substantial negative regulatory impact ofLuNAC61on flax fiber secondary wall formation. These results were consistent with the finding thatANAC012overexpression significantly reduces the cellulose content in stems (Koet al.2007).

4.2.Upstream and downstream regulatory networks of NAC genes affecting fiber development

Fig. 6 Subcellular localization analysis of the 35S:LuPLATZ24-GFP,35S:LuNAC61-GFP,and 35S:LuCesA10-GFP proteins in tobacco cells and verification of in vivo protein interactions. A,subcellular localization. B,dual-luciferase reporter assay.

NAC transcription factors influence various plant traits by regulating downstream gene expression and co-regulating other transcription factors. Although the ATrich sequence and zinc-binding (PLATZ)transcription factor family in plants is small(Naganoet al.2001),it plays a significant role in plant development and growth,and is regulated by ABA (Liet al.2017;Zhang Set al.2018;Wanget al.2019). The PLATZ family is present in several plant species,such as oilseed rape,olive,corn,wheat,and others (Wanget al.2018;Azimet al.2020),contributing to plant stress responses and developmental regulation.GmPLATZ negatively affects soybean drought tolerance,underscoring the role of PLATZ transcription factors in plant drought sensitivity (Zhaoet al.2022). AtPLATZ2 inhibits transcription upstream ofCBL4/SOS3andCBL10/SCaBP8,negatively regulatingArabidopsissalt tolerance (Liuet al.2020). Guoet al.(2022) explored how the bHLH transcription factorPGS1regulates PLATZ transcription factorFl3,thereby affecting grain seed size and weight. PLATZ transcription factor research has predominantly focused on stress responses,growth,and development,with limited reports on fiber development.Some scholars observed downregulated expression of certain NAC and PLATZ family members during cotton boll growth and development,indicating that these two types of transcription factors may play a role in coregulating fiber development during cotton growth and development(Handeet al.2017). In this study,LuPLATZ(LuPLATZ24)was found to be significantly coexpressed withLuNAC61,suggesting that this PLATZ transcription factor may be the upstream regulator ofLuNAC61,which is closely related to flax fiber development.

Cellulose is a key component of the cell wall that is critical for plant morphogenesis and growth and relies on cellulose synthase (CesA) as its key enzyme. Various CesA proteins serve distinct roles inArabidopsis. Notably,AtCesA1,AtCesA3,andAtCesA6contribute to primary cell wall formation,whileAtCesA4,AtCesA7,andAtCesA8are involved in SCW development (Pagantet al.2002;Szyjanowiczet al.2004). In particular,AtCesA1andAtCesA3play vital roles inArabidopsisdevelopment and growth (Perssonet al.2007). TheTaCesA,TaCOMTandTaCADgenes maybe enhance lodging resistance in wheat plants by improving the biosynthesis and accumulation of lignin and cellulose (Donget al.2023). Tanakaet al.(2003)observed that rice mutants created by insertingOsCesA4,OsCesA7,andOsCesA9into the retrotransposonTos17displayed brittle stems,reduced cellulose content,and thinner cortical fiber cell walls. This indicated that these functions are non-redundant and serve a common purpose.The secondary wall of flax fiber is mainly composed and thickened by cell synthesis and accumulation. The flax fiber secondary wall contains >90% cellulose,with cellulose synthesis mainly occurring on the plant cell wall protoplast membrane. Plant fibers are produced through a structured protein complex involving CesAs and other cellulose synthesis-related proteins. The subcellular localization of these three CesA proteins revealed their presence in the nucleus. The Dual-Luc experiments revealed the upregulation ofLuNAC61expression byLuPLATZ24,subsequently inhibitingLuCesA10expression,indicating an interaction between them.

To understand their regulatory interaction in flax fiber development,subcellular localization unveiled the presence of LuCesA10 in the endoplasmic reticulum and the nucleus. The rough endoplasmic reticulum is connected to the cell nuclear membrane,which indicates that the CesA protein moves between the nucleus and the rough endoplasmic reticulum. Furthermore,the CesA subunit needs to be assembled in the endoplasmic reticulum into a cellulose synthase complex (CSC),which is related to the primary wall formation,and then through the vesicular transport pathway to synthesize cellulose in the cytoplasmic membrane (McFarlaneet al.2014). Therefore,we speculate that whenLuNAC61is overexpressed in flax plants,it stimulatesLuPLATZ24expression accordingly. However,theLuCesA10expression level is consequently significantly inhibited,resulting in blocked LuCesA10 protein translation and a significant reduction in its quantity. Therefore,this significantly reduces the number of CSCs involved in fiber formation,ultimately negatively regulating fiber development in flax stems.

5.Conclusion

In this study,an evolutionary analysis of the LuNAC family indicated that its 17 members may regulate flax fiber development. Subsequent qRT-PCR screening functionally verifiedLuNAC61. OverexpressingLuNAC61in flax resulted in a thinner plant compared to the WT.Observable effects included a scarcity of young leaves in the top meristem,yellowing at the lower leaf tip,and reduced cellulose content and fiber bundles in the stem.GUS staining analysis revealed the widespread activity of theLuNAC61promoter across multiple flax tissues,with the highest activity found in the phloem fibers of the stems. This study identifiedLuPLATZ24as an upstream regulator andLuCesA6,LuCesA10,LuCesA20,LuCesA29,LuCesA35,andLuCesA40as genes significantly coexpressed withLuNAC61. Subcellular localization analysis indicated the nucleus and cytoplasm as the locations of the LuPLATZ24 protein,the nucleus as the location of the LuNAC61 protein,and the nucleus and endoplasmic reticulum as the locations of LuCesA10.LuPLATZ24was found to positively regulateLuNAC61,whileLuNAC61negatively regulateLuCesA10.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31801409),the Safe Preservation and Accurate Identification of Flax Germplasm Resources in South,China (23ZH174),the Construction of Modern Agricultural Industrial Technology System,China(CARS-16-E01),the Protection and Utilization of Crop Germplasm Resources,China (2016NWB044),and the National Science and Technology Resource Sharing Service Platform Project,China (NCGRC-2020-15).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.12.029