Yu Guo,Yumei Hu,Hun Chen,Pengshui Yn,Qingguo Du,Yfei Wng,Hongqiu Wng,Zhonghu Wng,Dingming Kng,Wen-Xue Li,*

a Key Laboratory of Crop Heterosis and Utilization,Ministry of Education,College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China

b National Engineering Laboratory for Crop Molecular Breeding,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:

A B S T R A C T Lodging is a major problem limiting maize yield worldwide.However,the mechanisms of lodging resistance remain incompletely understood for maize.Here,we evaluated 443 maize accessions for lodging resistance in the field.Five lodging-resistant accessions and five lodging-sensitive accessions were selected for further research.The leaf number,plant height,stem diameter,and rind penetrometer resistance were similar between lodging-resistant and-sensitive inbred lines.The average thickness of sclerenchymatous hypodermis layer was thicker and the vascular area was larger in the lodging-resistant lines compared with lodging-sensitive lines.Although total lignin content in stem tissue did not significantly differ between lodging-resistant and-sensitive lines,phloroglucinol staining revealed that the lignin content of the cell wall in the stem cortex and in the stem vascular tissue near the cortex was higher in the lodging-resistant lines than in the lodging-sensitive lines.Analysis of strand-specific RNA-seq transcriptome showed that a total of 793 genes were up-regulated and 713 genes were down-regulated in lodging-resistant lines relative to lodging-sensitive lines.The up-regulated genes in lodging-resistant lines were enriched in cell wall biogenesis.These results indicated that modification of cell wall biosynthesis would contribute to lodging resistance of maize.

1.Introduction

Lodging is one of the major factors limiting cereal productivity worldwide[1,2].By affecting dry matter assimilation and cereal harvest,lodging can reduce yield by up to 80%[3].Lodging can be defined as the displacement of cereal stems from their vertical position,and can be divided into stem lodging and root lodging.Whereas stem lodging refers to the buckling of the stalk/stem to the ground,root lodging is the inability of the roots to provide anchorage[4–6].Mechanical harvesting of lodged crops is difficult and usually leads to yield losses[7].

Maize ranks first in total production among major staple cereals,and high maize yield is needed to meet the growing requirements for biofuel and many other industrial products[8].With climate change,however,the frequency of extreme weather and therefore lodging is increasing in the main maize production area of China.In 2020,Typhoon Maysak resulted in the stem lodging of a large area of maize,especially in Northeast China.Stem lodging constitutes＞60% of the lodging in densely cultivated maize fields[9]and results in annual losses of Chinese maize production as high as 1 Mt[10].As a consequence,the development of lodging-resistant varieties has become a major goal of modern maize breeding,and this development depends on the identification of lodging-resistant maize inbred lines.Large-scale evaluation of maize germplasm for lodging resistance is therefore required.

Lodging is affected by both environmental factors and plant characteristics.Environmental factors included wind,rain,diseases,and insect pests.Among plant characteristics,plant height greatly affects the lodging resistance of crops[11,12].Since the 1960s,the introduction of semi-dwarf varieties has significantly increased grain yields by increasing lodging resistance,especially of rice and wheat[2].If maize plants are dwarf,however,ventilation and light penetration become inadequate,and grain yields are reduced.Researchers have previously reported that stem lodging resistance of maize is determined by the diameter and length of the stem’s basal internode[13],the section modulus of the stem[14],and ear height[15].The thickness of stem cell walls,the total area of vascular bundles,the vascular bundle area in the peripheral layer,and the auxiliary axis diameter of the stem in cross section also affect stem lodging resistance of maize[16–18].However,most findings on how morphological and anatomical characteristics affect stem lodging of maize have been obtained under laboratory conditions.How morphological and anatomical characteristics affect stem lodging of maize under field conditions has not been extensively studied.

Researchers also tried to understand the molecular mechanisms regulating stem lodging resistance of maize[19,20].Based on two recombinant inbred line population,qRPR3-1for rind penetrometer resistance was identified,and four genes associated with the biosynthesis of cell wall components were located in the quantitative trait loci[21].ZmNST3(NAC secondary wall thickening promoting factor3)was reported to improve maize lodging resistance by directly regulating the mRNA abundance of genes related to cell wall biosynthesis[22].Sequence amplified insertion flanking fragments and transgenic analysis revealed thatbrittle stalk4encoded a chitinase-like protein1(Ctl1),and overexpression ofZmCtl1enhanced mechanical stalk strength without affecting plant stature[12].Our group reported that miRNA528 affected lodging resistance of maize by regulating lignin biosynthesis under nitrogen-luxury conditions,and knockdown of ZmmiR528 or overexpression ofZmLAC3significantly increased the lignin content and rind penetrometer resistance of maize stems[23].However,the related conclusions were based on transgenic plants of single gene,and the molecular mechanisms regulating stem lodging resistance of maize were still far beyond understanding.

To better understand stem lodging resistance of maize,we evaluated 443 maize accessions under field conditions in the current study.After a storm that resulted in extensive lodging,five lodging-resistant accessions and five lodging-sensitive accessions were selected for further research.We found that the average thickness of sclerenchymatous hypodermis layer was thicker and the vascular area was larger in the lodging-resistant line than in the lodging-sensitive lines.Phloroglucinol staining showed that the lignin content in the cell wall of the cortex and of the stem vascular tissue near the cortex was lower in the lodging-sensitive lines than in the lodging-resistant lines.We further conducted a transcriptome analysis to identify the underling molecular processes involved in lodging resistance.Our results suggest that cell wall synthesis and modification may contribute to lodging resistance in maize.

2.Materials and methods

2.1.Plant materials and experimental setup

A total of 443 maize accessions were evaluated for lodgingstress tolerance under natural conditions.The accessions consisted of introgression lines,Chinese elite inbred lines,and inbred lines from different ecological zones in China,International Maize and Wheat Improvement Center(CIMMYT),and USA.The accessions included 384 temperate inbred lines and 59 tropical/subtropical inbred lines(Table S1).

The evaluation was conducted at the Shunyi Agriculture Experimental Station of Institute of Crop Sciences,Chinese Academy of Agricultural Sciences(40.13°N,116.65°E).The experimental site had an alkaline(pH 8.2)cinnamon soil that contained the following components per kg of dry soil:18.8 g of organic matter,85.79 mg of hydrolyzable nitrogen(N),40.7 mg of Olsenphosphorus(P),and 151.2 mg of exchangeable potassium(K).Before the seeds of the inbred lines were sown,750 kg ha-1of a compound fertilizer(15–15–15 N-P2O5-K2O)was uniformly broadcast.At the joint stage of maize,a topdressing of 450 kg ha-1of urea fertilizer(N≥46.4%)was applied.The maize accessions were evaluated in an alpha(0,1)lattice design[24].Seeds were sown on May 13,2020.Each line was sown in a two-row plot,with 10 plants per row,and with 25 cm between plants and 60 cm between rows.Irrigation was carried out on May 20 and June 20 with each irrigation being 70 mm.As indicated in the Results,a storm that occurred in the afternoon of July 1,2020 caused substantial stem lodging.

2.2.Selection of lodging-resistant and-sensitive genotypes under natural conditions

After this storm,we selected five lines that had not lodged and five lines that had lodged as representatives of lodging-resistant and lodging-sensitive lines,respectively.The evaluation of lodging resistance was based on stalk angle relative to the ground.The stalk angle of extremely poor resistance,poor resistance,moderate resistance,resistance and strong resistance was 0°–30°,30°–45°,45°–60°,60°– 75°,and 75°–90°,respectively.

2.3.Morphological traits of lodging-resistant and lodging-sensitive genotypes

The following morphological traits were assessed for the five lodging-resistant lines and the five lodging-sensitive lines:leaf number,plant height,stem diameter,and rind penetrometer resistance.The latter two properties were measured in the third internode from the bottom of the stem where breakage commonly occurred on lodging-sensitive lines.

2.4.Section and anatomical feature analysis

The third internode from the bottom of the stem was sampled from the five lodging-resistant and five lodging-sensitive lines.Four biological replicates were performed for each line.The samples were embedded in 3%agar for observation of cells and tissues via light microscopy.Transverse sections(50 μm)were produced using a vibratome(Leica VT 1000 S).The images were collected with a Leica DM6000M microscope.ImageJ software was used to measure the thickness of sclerenchymatous hypodermis layer and the area of vascular bundles.

As in the previous section,the part of the third internode from the bottom was sampled for two lodging-resistant lines(LI68 and CX77)and two lodging-sensitive lines(Q1261 and 14HF1994).The samples were cut into small squares and fixed overnight in 2.5%glutaraldehyde at 4 °C.After the samples were rinsed with 0.1 mol L-1PBS(pH 7.0)and re-fixed in 1% osmic acid,they were dehydrated in a graded series of ethanol.The samples were finally dried with a critical point dryer(Leica EM CPD300)and were sputter coated with gold palladium.The electron tomograms were collected with a Hitachi Regulus 8100 scanning electron microscope(Hitachi High Technologies,Tokyo,Japan)operating at 4.0 kV.

The vascular structure of the third internode from the bottom of the lodging-sensitive and lodging-resistant lines was visualized by Wiesner staining as previously described[25].Lignin appeared as an orange/red color under white light.The images were collected with Leica DM6000M light microscope.

2.5.Measurement of rind penetrometer resistance

The rind penetrometer resistance of the third internode from the bottom of the five lodging-resistant and five lodgingsensitive lines was determined as previously described[23].Three biological replicates were evaluated for each line.All samples were examined under the same conditions.

2.6.Determination of lignin,cellulose,and hemicellulose contents

The third internodes from the bottom of the lodging-resistant and lodging-sensitive lines were sampled to determine lignin,cellulose,and hemicellulose contents.These samples were dried and then milled into fine powders.Total lignin contents were determined by the AcBr method[26,27].Modified NREL procedures were used to determine the cellulose and hemicellulose contents[28].

2.7.RNA analysis

Stem tissue samples were collected from 3 representative plants per line 3 days after the storm,and total RNA was extracted from these samples using Trizol reagent(Invitrogen).The expression data should contain stress-responsive genes related to storm and injury.Real-time RT-PCR were performed as described by Sun et al.[23]with three biological replicates per line.The sequences of the primer pairs are listed in Table S2.

To gain insight into the molecular events involved in lodging resistance of maize,a total of 20 strand-specific RNA libraries from the third internode of lodging-resistant and lodgingsensitive lines were generated.Each line was represented by two biological replicates.The total RNA was extracted by Trans-Zol Up(TransGen Biotech,China),and the RNA quality and integrity were assessed using Aligent 2100.To generate the strand-specific sequencing libraries,a total amount of 3 μg RNA per sample was used to remove ribosomal RNA by Epicentre Ribo-ZeroTM rRNA Removal kit(Epicentre,USA)firstly.The libraries were generated by NEBNext Ultra Directional RNA Library Prep Kit for Illumina(NEB,USA)according to the manufacturer’s recommendations.First-strand cDNA was synthesized with random hexamers primer.After second-strand cDNA synthesis,terminal repair and ligation of poly(A)/sequencing oligonucleotide adaptors were carried out.Then,UNG enzyme was used to degrade the second strand of cDNA containing U.The fragments with expected size were purified and then amplified by PCR to generate the strand specific cDNA libraries.The libraries were sequenced with the Illumina Hiseq 2500 platform(Berry Genomics,Beijing,China).

The original raw reads were excluded low quality reads and adapter sequences using fastp[29].Reads that passed the filter were then aligned to the maize B73_RefGen_v4(ftp://ftp.ensemblgenomes.org/pub/plants/release-41/fasta/zea_mays/dna/)using HISAT2 v2.1.0[30].Only perfectly matching sequences were considered for further analysis.The count information was used to determine normalized gene expression levels as FPKM(Fragments Per Kilobase of transcript per Million mapped reads)[31].To identify the differentially expressed genes(DEGs),feature Counts software and R package‘‘edgeR”were used[32,33].Genes were considered as DEGs if the log2fold-change ratio was≥1 and if the adjustedPvalue was＜0.05.Gene ontology(GO)enrichment analyses were performed using AgriGO v2.0 with Maize AGPv4 as the reference background[34].

Fig.1.Selection of lodging-resistant(LR)and lodging-sensitive(LS)inbred lines under field conditions.(A)Precipitation and maximum wind speed of an unexpected storm that occurred at the experimental site;data were recorded by an on-site automatic weather station.(B)The responses of lodging-resistant and lodging-sensitive inbred lines to the storm.Numbers below the photographs indicate the rate of stem lodging,i.e.,the percentage of the total number of stems that lodged for the indicated line.

Fig.2.Leaf number(A),plant height(B),diameter of the third stem(C),and rind penetrometer resistance(D)of lodging-resistant(LR)and lodging-sensitive(LS)inbred lines.Thick lines within the boxes are medians,dashed lines indicate variability outside the upper and lower quartiles,and small open circles represent the individual values.‘‘ns”indicates no significant difference as determined by t-tests.

3.Results

3.1.Selection of lodging-resistant and lodging-sensitive genotypes under field conditions

The 443 maize accessions were originally evaluated for low nitrogen tolerance under field conditions.In the afternoon of July 1,2020,a violent rainstorm struck the area of the Shunyi Agriculture Experimental Station,Beijing.Total precipitation per hour was～50 mm,and the maximum wind speed exceeded 20 m s-1(Fig.1A).The extreme weather provided a good opportunity to evaluate the maize germplasm for lodging resistance under natural conditions[35].The stems of most inbred lines were damaged in either replicates(Table S1).None of the stems of inbred lines LI55,LI68,CX41,CX77,and DR-F became bent or broken during the rainstorm,and these five lines were therefore considered to be lodging-resistant(Fig.1B).In contrast,the stems of Q1261,Chuan273,14HF1994,JI495,and CML338 fractured during the rainstorm in both replicates,with breakage rates ranging from 20.0%to 42.9%(Fig.1B);these five lines were therefore considered to be lodging-sensitive.These lines originated independently[36].

3.2.Morphological traits of lodging-resistant and-sensitive genotypes

The maize plants of all 10 lines had 8–9 leaves(Fig.2A),indicating that the lodging-resistant and-sensitive lines were at a similar developmental stage.Plant height did not significantly differ among the lines,except that LI68 was shorter than the other lines(Figs.2B,S1A).The stem diameter and rind penetrometer resistance of the third stem from bottom were also similar between lodging-resistant and lodging-sensitive lines(Figs.2C and D,S1B).These results suggested that morphological traits were not the critical factors determining the lodging resistance of these maize lines.

3.3.Anatomical traits of lodging-resistant and-sensitive genotypes

Because the morphological traits of lodging-resistant and lodging-sensitive genotypes were similar,we hypothesized that anatomical traits might contribute to the lodging resistance of maize.To test this hypothesis,we examined the microstructure of the cortex and vascular tissue near the cortex.The average thickness of sclerenchymatous hypodermis layer in the lodgingresistant genotypes was 45.5 μm,which was 2 times greater than in the lodging-sensitive genotypes(Fig.3A and B).Among the lodging-resistant lines,the sclerenchymatous hypodermis layer was thinnest for DR-F(35.8 μm),but the sclerenchymatous hypodermis layer was 14% thicker for DR-F than for CML338,which had the thickest sclerenchymatous hypodermis layer among the lodging-sensitive lines(Fig.S2).We also observed the thickness of sclerenchymatous hypodermis in lodging-resistant andsensitive lines under greenhouse condition,and got similar results as those after the storm(Fig.S3).These results suggested that the thickness of sclerenchymatous hypodermis layer contributes to lodging resistance of maize.

Fig.3.Anatomical analysis of lodging-resistant(LR)and lodging-sensitive(LS)inbred lines by light microscopy.(A)Representative images of transections of the third stem from the bottom of the field-grown lines.Red lines indicate the cortical and vascular sclerenchyma cells,respectively.Scale bars=30 μm for cortical sclerenchyma,50 μm for vascular sclerenchyma,and 250 μm for vascular tissue.(B)The thickness of sclerenchymatous hypodermis layer and vascular sclerenchymatous cell layer.(C)The number and the area of vascular bundles near the cortex.Thick lines within the boxes are medians,dashed lines indicate variability outside the upper and lower quartiles,and small open circles represent the individual values.Asterisks indicate significant differences as determined by t-tests(*P＜0.05,**P＜0.01).

Fig.4.Scanning electron micrographs of transections of stem of lodging-resistant(LR)and lodging-sensitive(LS)lines.Scale bars,100 μm.CS,cortical sclerenchyma cells;VS,vascular sclerenchyma cells;VC,vascular cylinder;MX,metaxylem;PX,protoxylem;P,phloem.

We also examined the sclerenchyma bundle sheaths in the vascular tissue near the cortex.The average thickness of vascular sclerenchymatous cell layer was also significantly greater in the lodging-resistant lines than in the lodging-sensitive lines(Fig.3A and B).However,not all of lodging-sensitive lines had thin vascular sclerenchymatous cell layer(Fig.S2).The thickness of vascular sclerenchymatous cell layer in the lodging-sensitive lines Ji495 and CML338 was similar to that in the lodging-resistant lines(Fig.S2B).The rind region of the lodging-resistant lines contained fewer but larger vascular bundles than the rind region of the lodging-sensitive lines(Figs.3C,S4).The average vascular area was 2 times greater in the lodging-resistant lines than in the lodging-sensitive lines(Fig.3C).This suggested that the size of the vascular area near the cortex also affected the lodging resistance of maize.

To verify the results obtained by light microscopy,we used scanning electron microscopy to examine the stem tissue of two lodging-resistant lines(LI68 and CX77)and two lodging-sensitive lines(Q1261 and 14HF1994).The arrangement of the cortical sclerenchyma cells was much denser in the lodging-resistant lines than in the lodging-sensitive lines(Fig.4).The cell walls of sclerenchyma cells near the cortex and vascular tissue were also thinner in the lodging-sensitive lines than in the lodging-resistant inbred lines(Fig.4).

3.4.Stem chemical composition of lodging-resistant and-sensitive genotypes

Lignin is a phenylpropanoid-derived polymer that is specifically deposited in secondary cell walls where it increases stem strength[37,38].When we used acetyl bromide(AcBr)analysis[26]to quantify the total lignin content of the third internode from the bottom of the stem,we found that total lignin content did not significantly differ between lodging-resistant and lodging-sensitive lines(Fig.5A).We also measured cellulose and hemicellulose contents,and found that the lodging-resistant and-sensitive lines contained similar cellulose and hemicellulose contents(Fig.5A).This indicated that lodging resistance was not affected by cellulose or hemicellulose accumulation in the maize lines.

Considering that the cortical sclerenchyma cell walls were thinner in the lodging-sensitive lines than in the lodging-resistant lines,we hypothesized that lignin accumulation in the cortical sclerenchyma tissue would be greater in the lodging-resistant lines than in the lodging-sensitive lines.To test the hypothesis,we stained the stem tissues with phloroglucinol,which is a ligninspecific indicator of secondary wall thickening[25].The staining of the cortex and of the vascular tissue near the cortex of stems indicated that lignin content was lower in lodging-sensitive lines than in the lodging-resistant lines(Fig.5B and C).

3.5.Strand-specific RNA-Seq transcriptome analysis of lodgingresistant and-sensitive genotypes

The RNA libraries yielded a total of～800 million raw reads,and approximately 97% of the raw reads remained after removal of adaptors,Ns reads,and low quantity reads.Of these,approximately 86% could be mapped to the maize B73 v4 reference genome(ftp://ftp.ensemblgenomes.org/pub/plants/release-41/fasta/zea_mays/dna/).Sequences that could not be mapped to the maize genome were discarded,and only those that perfectly mapped were analyzed further.Overall,a total of 320 million reads from the lodging-resistant library and 340 million reads from the lodging-sensitive library were retained for further research.

Fig.5.Chemical composition of stems of lodging-resistant(LR)and lodging-sensitive(LS)lines.(A)Contents of AcBr lignin,cellulose,and hemicellulose in stems.DW represents dry weight.Thick lines within the boxes are medians,dashed lines indicate variability outside the upper and lower quartiles,and small open circles represent the individual values.‘‘ns”indicates no significant differences as determined by t-tests.(B)Phloroglucinol staining of lignin in stems.Scale bars,100 μm.(C)The staining signal intensity of sclerenchymatous hypodermis layer and vascular sclerenchymatous cell layer.Relative staining intensity was represented by the gray values calculated by ImageJ.Thick lines within the boxes are medians,dashed lines indicate variability outside the upper and lower quartiles,and small open circles represent the individual values.Asterisks indicate significant differences as determined by t-tests(*P＜0.05,**P＜0.01).

Pearson’s correlation coefficients were calculated to determine the relationships between the biological replicates of each line.Although the RNA was obtained from field-grown maize,thervalues were high(～0.9)except for 14HF1994.The two RNA libraries from 14HF1994 were not analyzed further due to their low Pearson’s correlation coefficient.Based on the criteria that the log2foldchange ratio was≥1 and that the adjustedPvalue was≤0.05,1506 genes were identified by DEGseq software as DEGs between resistant and sensitive lines[39].Among the DEGs,793 genes were up-regulated and 713 genes were down-regulated in lodgingresistant lines relative to lodging-sensitive lines(Fig.6A;Table S3).The expression profiles of selected genes determined by qRT-PCR were consistent with our RNA-seq data(Fig.S6),indicating that our RNA-seq data were reliable.We then performed Gene Ontology(GO;http://bioinfo.cau.edu.cn/agriGO/)analysis to determine the molecular events related to the DEGs.The upregulated genes in the third internode from bottom of lodgingresistant lines were mainly enriched in cell wall biogenesis(GO:0042546),plant-type cell wall biogenesis(GO:0009832),planttype primary cell wall biogenesis(GO:0009833),phenylpropanoid metabolic process(GO:0009698),phenylpropanoid biosynthetic process(GO:0009699),cellulose synthase(UDP-forming)activity(GO:0009699),and secondary metabolite biosynthetic process(GO:0044550)(Fig.6B).The down-regulated genes in the third internode from bottom of lodging-resistant lines were mainly enriched in cellular response to starvation(GO:0009267),iron ion homeostasis(GO:0055072),fatty acid beta-oxidation(GO:0006635),and calcium-dependent protein serine/threonine kinase activity(GO:0009931)(Fig.6C).

3.6.DEGs were associated with cell wall biosynthesis

The up-regulated genes in the third internode from bottom of lodging-resistant lines were mainly enriched in cell wall biogenesis.This prompted us to focus on genes involved in cell wall biosynthesis.We only considered the genes that the FPKM exceeded 1 in at least one of the libraries and log2fold-change ratio was≥2.A total of 14 genes,including MYB,WRKY,OFP17,NAC,laccase,and UDP-glycosyltransferase(Table 1)were identified.We randomly chose two DEGs listed in Table 1(Zm00001d040621 and Zm00001d044409)and validated their difference in expression levels in lodging-resistant lines versus lodging-resistant lines by RT-qPCR(Fig.S7).These results indicated the importance of cell wall biosynthesis in the lodging resistance of maize.

Fig.6.Differentially expressed genes between lodging-resistant(LR)and lodging-sensitive(LS)lines.(A)Global view of differentially expressed genes(DEGs)between LR and LS lines.(B)Gene Ontology(GO)classifications of up-regulated DEGs with fold enrichment exceeding 2 in LR lines compared with LS lines.(C)GO classifications of downregulated DEGs with fold enrichment exceeding 2 in LR lines compared with LS lines.The point size represents the number of genes in the pathway;the color depth means-log10(P-value).

Table 1Differentially expressed genes involved in cell wall biosynthesis(log2 fold-change≥2).

4.Discussion

In the present research,we evaluated 443 maize accessions for lodging resistance under field conditions.Based on the physiological indices tested,we found that the thickness of sclerenchymatous hypodermis layer plays an important role in lodging resistance of maize.Based on analysis of strand-specific RNA-seq transcriptome,we hypothesized that the modification of cell wall biosynthesis contributes to lodging resistance of maize.

One challenge in developing lodging-resistant maize varieties is the large-scale testing of lodging propensity of multiple inbred lines under field conditions.Some results have demonstrated that stalk strength might be a reliable predictor of lodging propensity[14,40].Several methods such as rind penetrometer resistance(RPR),a three-point bend test,and computed tomography(CT)scanning have been developed to quantify the stalk strength of maize.The three-point bending test and CT scanning,however,are time consuming and expensive[41].RPR is rapid and could be used to assess stalk strength of maize on a large scale[42].However,PRP is affected by the shape and size of the penetrating probe and did not create stronger stalks[43,44].Consistent with these observations,we found that the PRR as well as plant height and stem diameter did not differ between lodging-resistant and lodging-sensitive lines.Compared with the five lodging-sensitive lines,all five lodging-resistant lines had cortical sclerenchyma cells with thicker layers.It’s worth noting that the anatomical and historical observations were performed several days after storm.The morphological traits of lodging-resistant and-sensitive genotypes grown in the greenhouse without stress were also examined,and we obtained similar results to those with stress in the field.These results suggested that the thickness of sclerenchymatous hypodermis layer could be used to predict stem lodging resistance of maize.

It is reasonable to suspect that lodging resistance in cereals is related to the chemical composition of the stem,including its cellulose,hemicellulose,and lignin contents.Lignin is only deposited in secondary cell wall that affected structural integrity of the cell wall and stiffness of the stem[37,38,45].The lignin content in stems was significantly correlated with stem strength and lodging resistance in wheat[5,46].In the current study,we found that the total lignin content of the third stem from bottom was similar between lodging-resistant and lodging-sensitive lines.This might be due to the higher vascular number in the lodging-sensitive lines than in the lodging-resistant lines.Using the lignin-specific phloroglucinol stain,however,we found that the lignin content in the stem cortex and the stem vascular tissue near the cortex was higher in lodging-resistant lines than in lodging-sensitive lines.The genes encoding phenylalanine ammonia lyase(PAL)and laccase were up-regulated in lodging-resistant lines.PAL catalyzes the first step in a series of enzymatic reactions generating lignin monolignols from phenylalanine[47].Our previous results also indicated that the lodging propensity of maize could be changed by regulating lignin biosynthesis[23].These results suggest that maize lodging resistance might be increased by regulating lignin biosynthesis in specific cells.

Lignin biosynthesis can be altered by modifying the expression of a transcription factor that altered the mRNA abundance of downstream target genes.InArabidopsis,overexpression ofMYB58andMYB63specifically activates lignin biosynthetic genes and concomitant ectopic deposition of lignin in cells that are normally unlignified[48].In contrast,a loss-of-function mutation inMYB75upregulated the expression level of genes associated with lignin biosynthesis and cellulose[49].Mutation of WRKY transcription factors increased the biosynthesis of xylan,cellulose,and lignin required for secondary wall thickening and consequently increased stem biomass by up to 50%[50].NAC domain transcription factor is a key regulator of secondary wall synthesis in fibers ofArabidopsis[51].Ovate Family Protein(OFP)transcription co-regulators could interact with KNAT7 to regulate secondary cell wall formation inArabidopsis[52].Our analysis of strand-specific RNA-Seq transcriptome identified 7 MYBs,2 NACs,1 WRKY and 1 OFP as DEGs that log2fold-change was≥2.Though the functions of these genes in maize require further research,they should be the targets for the development of lodging stress-tolerant maize lines.

Increased researches were focused on the analysis of RNA-seq data produced from a collection of accessions[53,54].In present study,five plants with similar growth stage were sampled as one biological replicate,and each accession represented by two biological replicates was used for RNA-library construction and sequencing.The average expression correlation coefficient of the biological replicates was more than 0.88,indicating a reasonable reproducibility of the extensive sampling.The reads count of lodging-resistant and-sensitive lines was averaged,respectively.The edgeR package was used to identify DEGs[32,33].This analysis would improve insights into the biological networks and molecular pathways of lodging-resistance in maize.It needs to be pointed out that the gene expression data were collected from the plants three days after the storm.Some of the changes in gene expression may be associated with stress response of these genotypes.Thus,caution needs to be taken to interpret DEGs in this study.

CRediT authorship contribution statement

Yu Guo:Formal analysis,Investigation,Methodology,Visualization,Writing-original draft.Yumei Hu:Investigation,Supervision,Validation.Huan Chen:Investigation,Supervision,Visualization.Pengshuai Yan:Investigation,Visualization.Qingguo Du:Investigation,Software.Yafei Wang:Investigation.Hongqiu Wang:Investigation.Zhonghua Wang:Investigation.Dingming Kang:Visualization.Wen-Xue Lidesigned the research and wrote the article:Conceptualization,Data curation,Funding acquisition,Resources,Writing-review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China(31861143004),the National Key Research and Development Program of China(2016YFD0100701),and the Agricultural Science and Technology Innovation Program of CAAS to WXL.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.01.002.