Jipeng Xing, Yubin Wng,b, Qingqing Yo, Yushi Zhng, Mingci Zhng,*, Zhohu Li
a State Key Laboratory of Plant Physiology and Biochemistry, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
b Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
Keywords:Brassinosteroids Nitrogen uptake Nitrate transporter gene Root architecture Maize
ABSTRACT Brassinosteroids(BRs)are steroid hormones that function in plant growth and development and response to environmental stresses and nutrient supplies.However,few studies have investigated the effect of BRs in modulating the physiological response to nitrogen(N)supply in maize.In the present study,BR signalingdeficient mutant zmbri1-RNAi lines and exogenous application of 2,4-epibrassinolide (eBL) were used to study the role of BRs in the regulation of physiological response in maize seedlings supplied with N.Exogenous application of eBL increased primary root length and plant biomass,but zmbri1 plants showed shorter primary roots and less plant biomass than wild-type plants under low N(LN)and normal N(NN)conditions.LN induced the expression of the BR signaling-associated genes ZmDWF4, ZmCPD, ZmDET2,and ZmBZR1 and the production of longer primary roots than NN.Knockdown of ZmBRI1 weakened the biological effects of LN-induced primary root elongation.eBL treatment increased N accumulation in shoots and roots of maize seedlings exposed to LN or NN treatment.Correspondingly,zmbri1 plants showed lower N accumulation in shoots and roots than wild-type plants.Along with reduced N accumulation, zmbri1 plants showed lower fluxes and 15 uptake.The expression of nitrate transporter (NRT) genes(ZmNPF6.4, ZmNPF6.6, ZmNRT2.1, ZmNRT2.2) was lower in zmbri1 than in wild-type roots, but eBL treatments up-regulated the transcript expression of NRT genes.Thus,BRs modulated N physiological response and regulated the transcript expression of NRT genes to promote N uptake in maize.
Brassinosteroids (BRs) are plant-specific steroidal hormones involved in plant growth and developmental processes and response to various abiotic stresses.Studies have revealed BRs influence root architecture, which determinies the efficiency of water and nutrient acquisition in higher plants.High concentrations of BRs inhibit, and low concentrations promote, root growth[1,2].InArabidopsis,the BR-insensitive mutantsbri1-116andbes1-D(gain-of-function mutants) exhibit short roots [3,4].The root elongation ofzmbri1-RNAi plants is insensitive to brassinolide treatment [5].Root development is a highly plastic process that is sensitive to nutrient limitation and other environmental parameters, with plant hormones acting as signaling mechanisms [6,7].BRs are involved in nitrogen (N)-modulated root elongation processes inArabidopsis.In a whole-genome association analysis,BRASSINOSTEROID SIGNALING KINASE3 (BSK3) functioned in root elongation response under low N condition[8].The BR biosynthesis geneAtDWF1is also a key gene involved in the root elongation response induced by N deficiency,and its overexpression increased N accumulation [9].These findings support the role of BRs in root development modulated by nutrition absorption.However, the function of BRs in N-modulated root architecture development in crops, especially monocots, has been little studied.
Recent research has focused on the role of BRs in plant growth and development adapted to nutrient-deficiency stress [10].In rice, iron (Fe) deficiency inhibits BR biosynthesis, and reduced endogenous BRs contribute to Fe transport and translocation from roots to shoots [11].Besides their involvement in root development in response to N deficiency,recent studies [12,13] have suggested that BRs are also involved in N uptake and metabolism processes.BRs regulate ammonium () uptake via regulation oftransporters inArabidopsisand rice.BR signaling acts in tomato plant response to N starvation by regulating autophagy[14].In cucumber, exogenous application of 2,4-epibrassinolide(eBL)reduced the harmful effects of suboptimal root zone temperature by changing the N metabolism and the flux rates ofand nitrate (), thereby increasing the growth of seedlings [15].These studies suggest positive roles of BRs in plant N uptake.
Genetic and breeding studies in maize,a major food and industrial crop, have shown that BRs influence plant architecture and kernel size regulation,thereby contributing to increase grain yield.Maize BR-deficient mutants have shortened internodes, twisted,dark green, erect leaves, and feminized male flowers [5,31,32].
ZmRAVL1regulatesbrd1(brassinosteroid C-6 oxidase1) to alter endogenous BR content.ZmRAVL1-edited lines displayed reduced leaf angle and compacted plant architecture leading to increased plant density and potential yield [33].ZmBES1/BZR1-5 positively regulates maize kernel size [34].
In maize production,external N inputs and their soil availability are the main nutrient-limiting factors in yield,and increasing N use efficiency is a goal of production[35].Identifying the physiological and molecular mechanisms of N uptake and utilization would further the breeding of N-efficient maize cultivars.N supply mediated the biosynthesis of BRs to modulate root elongation inArabidopsis[9].Overexpression ofZmDWF4increased grain yield by enhancing photosynthetic ability,promoting the expression of genes regulating cell division and the storage reservoir in maize kernels [36].Exogenous application of BR regulated maize growth and biomass accumulation to increase yield under either stress or normal field condition [37,38].The balance between carbon and N is the basis of high yield in crop production, but little is known about how BRs modulateuptake in maize plants in response to N supply.
The aim of this study was to investigate the role of BRs in the regulation of physiological responses to N supply in maize.The BR signaling-deficient mutantzmbri1-RNAi and exogenous eBL treatment were used for measuring the N uptake under low N(LN) and normal N (NN) conditions.A noninvasive micro-test(NMT) and15N labeling were used for evaluating BR-mediateduptake.
ZmBRI1RNA interference(RNAi)plants were obtained from the Genetics,Development,and Cell Biology Department of Iowa State University [5].The gene expression ofZmBRI1was decreased inzmbri1plants (Fig.S1).Seeds of wild-type (WT) maize (Zea maysL.cv.B73) andzmbri1plants were surface sterilized in a 10% (v/v) H2O2solution for 20 min and germinated on sand for 7 days in a growth chamber, at 28/22 °C with a 16 h/8 h light/dark cycle and 70%-80%relative humidity.Uniform seedlings with two visible leaves were transferred to half-strength culture solution for 2 days and then to full-strength solution (0.5 mmol L-1MgSO4,0.1 mmol L-1KH2PO4, 1 mmol L-1CaCl2, 0.1 mmol L-1EDTA-Fe,0.03 mmol L-1H3BO3, 0.0025 mmol L-1ZnSO4, 0.008 mmol L-1CuSO4, 0.005 mmol L-1MnSO4,and 0.0003 mmol L-1(NH4)6Mo7-O24),but supplied with differentconcentrations(2.0 mmol L-1KNO3,NN;0.05 mmol L-1KNO3,LN).The concentration of K+in the LN solution was supplemented with KCl to the same level as that in the NN solution.The nutrient solution was renewed every 3 days.When seedlings were transferred either the NN or the LN solution,treatment with 2,4-epibrassinolide (eBL) (RealTimes, CAS:78821-43-9) was applied simultaneously.The eBL was first dissolved in ethanol to make a stock liquor and then added into nutrient solution to a final concentration of 0.05 nmol L-1at every change of solution.The same dose of ethanol was added to the control.
Uniform seedlings from each treatment were harvested and separated into roots and shoots 6 days after NN and LN treatments.Primary root length was measured with a ruler.The relative rate of primary root length induced by LN is calculated by (LN - NN)/LN.Total root length and surface area were determined with WinRHIZO software (Pro 2014b, Beijing, China) from images acquired with an Epson V700 (Seiko Epson Corp, Nagano-ken, Japan) scanner.Samples for measuring plant biomass were heated for 30 min at 105 °C and dried for 3 days at 75 °C and dry weight was recorded using a electronic balance.Ten biological replicates were used for each treatment.
After LN or NN treatment for 5 days, uniform seedlings were selected for15N labeling.Roots were rinsed with 0.1 mmol L-1CaSO4solution for 1 min and transferred into nutrient solution with 0.05 or 2 mmol L-1K15NO3with a 99% atom excess of15N for 10 min.They were then rinsed for 1 min in 0.1 mmol L-1CaSO4solution before sampling.Samples were harvested separately and oven-dried for 3 days at 75 °C to constant weight.Dry samples were weighed and ground.The powder was used for total15N determination by isotope ratio mass spectrometry (Vario PYRO cube ISOprime 100, Isoprime/Elementar Ltd., Cheadle Hulme,UK).Total N accumulation in roots and shoots was estimated following Bremner et al.[40].Plant total N accumulation was calculated as the product of N concentration and corresponding dry weight.Three biological samples were used for each treatment.
Total RNA was isolated from each sample using an EASYspin rapid plant extraction kit(Aialab,Beijing,China),and reverse transcription was preformed using Oligo d (T) primer and M-MLV reverse transcriptase (Takara, Japan).The qRT-PCR was conducted in an Applied Biosystems 7500 Fast Real-Time PCR System(Applied Biosystems, USA) using TB Green Premix Ex Taq II (Takara, Japan).Gene expression was calibrated to the expression ofZmUBC(ubiquitin C).The relative gene expression was calculated by the 2-ΔΔCTmethod [41].Three biological replicates were used for each treatment.The primers for qRT-PCR are listed in Table S1.
Analysis of variance was performed using the general linear model (GLM) procedure in SPSS 21.0 (SPSS Inc., Chicago, IL, USA).Means of more than two groups were compared by Duncan’s multiple-range test atP<0.05.Changes in biomass and N accumulation were tested with Fisher’s LSD test atP< 0.05.
As shown in Fig.1a,LN inhibited the growth of roots and shoots compared to NN, while eBL treatment promoted shoot and root growth under both N conditions.The relative rate of primary root length induced by LN was 9.6% in eBL-treated plants and 7.3% in control plants(Fig.1b).In comparison with control plants,the total root length and root surface area increased by respectively 17.4%and 16.4% in eBL-treated plants under the NN condition and by 22.9% and 20.2% under the LN condition (Fig.S2).Shoot and root biomass increased by respectively 21.0% and 15.0% in eBL-treated plants under the NN condition and by 24.6% and 19.9% under the LN condition (Fig.1c, d).However, the ratio of root to shoot showed no significant difference between eBL-treated and control plants under LN and NN conditions (Fig.1e).
Thezmbri1plants showed a dwarf phenotype, lower primary root length, total root length, and root surface area than wildtype plants under both N conditions (Fig.2a).The relative rate of primary root length induced by LN was 1.2%-2.3%inzmbri1plants but 9.1% in wild-type plants (Fig.2b).In comparison with wildtype plants, total root length and root surface area decreased by respectively 50.6%-51.8% and 58.2%-60.8% inzmbri1plants under the NN condition but by 52.3%-52.5% and 59.4%-61.5% under the LN condition (Fig.S3).Shoot and root biomass decreased by 61.2%-63.8%and 54.2%-60.7%inzmbri1plants in comparison with wild-type plants under the NN condition, but by 62.1%-64.3% and 53.6%-61.2%under the LN condition(Fig.2c,d).The ratio of root to shoot was higher inzmbri1plants than in wild-type plants(Fig.2e).
As shown in Fig.3a, LN significantly induced the expression ofZmDWF4compared to NN at 1-6 days after N treatments.The expression levels ofZmCPDandZmDET2in LN-treated seedlings were also higher than those in NN-treated plants (Fig.3b, c).LN significantly increased the expression levels ofZmBZR1, a gene mediating pleiotropic BR responses, in comparison with NN at 1-6 days after N treatments (Fig.3d).
To determine the role of BRs involved in N uptake in maize seedlings, N accumulation was measured in eBL-treated plants and zmbri1plants under both N conditions.Under NN condition,eBL treatment significantly increased N accumulation in shoots and roots in comparison with control plants.N accumulation in shoots and roots in eBL-treated plants increased by respectively 11.6%and 31.5%under the LN condition(Fig.4a).N accumulations in shoots and roots inzmbri1plants were lower than those in wildtype plants under LN and NN conditions.They decreased by 49.8%-51.9% and 43.9%-48.1% under NN and by 57.3%-57.8% and 50.1%-53.1% under LN (Fig.4b).
To test whether the effects of BRs on N accumulation were due to plant growth or nutritional status,the dynamic accumulation of dry biomass and total N accumulation were measured in wild-type andzmbri1plants under LN and NN conditions.LN reduced the dry biomass of shoot and root compared to NN, whilezmbri1plants presented lower dry biomass of shoot and root than wild-type plants at 1-9 days after LN or NN treatment (Fig.5a, b).Although the dry biomass of shoot and root was lower inzmbri1plants than in wild-type plants under both LN and NN conditions,there was no significant difference between wild-type andzmbri1plants in the relative inhibition rate by LN to NN of dry biomass(Fig.5c,d).Similarly,zmbri1plants showed lower N accumulation in shoots and roots than wild-type plants at 1-9 days after LN or NN treatment(Fig.5e, f).However,zmbri1plants showed higher relative inhibition rates of LN in N accumulation in shoots and roots than wildtype plants at 1-9 days after N treatments (Fig.5g, h).
To clarify the function of BRs in modulating N uptake, netfluxes were measured by NMT in wild-type andzmbri1roots.As shown in Fig.6a, LN significantly repressed netfluxes in wild-type andzmbri1roots in comparison with NN, while netfluxes were markedly decreased inzmbri1roots relative to those in wild-type roots under LN and NN conditions.In comparison with wild-type roots, meanfluxes were decreased by 31.7%-45.9% inzmbri1roots under the NN condition and by 37.1%-57.5% inzmbri1roots under the LN condition (Fig.6b).The15N tracing assay suggested that the amount of15N inzmbri1plants was less than that in wild-type plants, while LN reduced the amount of15N in wild-type andzmbri1plants (Fig.6c).15fluxes also decreased inzmbri1plants (Fig.6d).
To investigate how BRs altered N uptake, the expression levels ofuptake-related genes were measured in eBL-treated,wild-type, andzmbri1roots responding to NN and LN.As shown in Fig.7a, the transcription levels ofZmNPF6.4were significantly decreased inzmbri1roots in comparison with wild-type roots,while LN down-regulated the expression ofZmNPF6.4in wildtype andzmbri1roots at 6 days after N treatments.Similarly toZmNPF6.4, the expression levels ofZmNPF6.6were lower inzmbri1roots than those in wild-type roots under both LN and NN conditions at 1-6 days after N supply (Fig.7b).Compared to NN, the expressions ofZmNRT2.1andZmNRT2.2were down-regulated at 3 and 6 days after LN treatment in wild-type andzmbri1roots(Fig.7c-d).The expression levels of these genes inzmbri1roots were lower than those in wild-type roots under LN and NN conditions.
Fig.1.Changes in morphology and plant biomass in control and eBL-treated plants in response to N supply.(a)Phenotypic characteristics of eBL-treated plants at 6 days after LN or NN.Scale bars,10 cm.(b-e)Effects of eBL on primary root length(b),shoot dry weight(c),root dry weight(d),and ratio of root to shoot(e)in maize seedlings under LN and NN conditions.The control was maize cv.B73.Values with error bars represent mean±SD(n=10).Different letters indicate significant difference between treatments according to Duncan’s multiple range test at P < 0.05.eBL, 2,4-epibrassinolide; LN, low N; NN, normal N.
Exogenous eBL application was used to further clarify the regulation by BRs of the transcript expression ofuptake-associated genes under both LN and NN conditions.eBL treatment significantly up-regulated the expression ofZmNPF6.4andZmNPF6.6relative to the control at 1-6 days after N supply (Fig.7e, f).eBLtreated plants showed higher transcript expression levels ofZmNRT2.1andZmNRT2.2than control plants under both LN and NN conditions (Fig.7g, h).
Brassinosteroids are growth-promoting steroidal hormones that play important roles in physiological and developmental adaptation to plants environment conditions [42-44].BRs are also involved in modulating the adaptation of plants in response to the supply of nutrients such as Fe and Pi [11,45].Several studies[10] have suggested that BRs play important roles in mediating N uptake and metabolism processes in plants.In the present study,exogenous eBL treatment and the BR signaling-deficient mutantzmbri1-RNAi affected the accumulation of shoot and root biomass,and the ratio of root to shoot in maize seedlings in response to LN or NN.These findings suggested that BR accumulation affects the adaptation of the maize plant to N supply in the soil.LN induced the expression of the BR signaling-associated genesZmDWF4,ZmCPD,ZmDET2,andZmBZR1and the production of longer primary roots than NN.Similarly,low N regulated the biosynthesis of BRs to stimulate root elongation inArabidopsis[9].Thus, BRs influenced the response to N supply in maize.
Fig.2.Changes in morphology and plant biomass in zmbri1 plants under LN and NN conditions.(a)Phenotypic characteristics of zmbri1 plants at 6 days after LN or NN.Scale bars, 10 cm.(b-e) Changes in primary root length (b), shoot dry weight (c), root dry weight (d), and ratio of root to shoot (e) in zmbri1 plants under LN and NN conditions.Values with error bars are mean±SD(n=10).Different letters indicate significant difference between treatments by Duncan’s multiple range test at P<0.05.WT,wild type;LN, low N; NN, normal N.
Fig.3.Effects of N supply on expression of ZmDWF4 (a),ZmCPD (b),ZmDET2(c), and ZmBZR1 (d) in maize (cv.B73)seedlings.Values with error bars are mean±SD(n=3).Different letters indicate significant difference between treatments by Duncan’s multiple range test at P < 0.05.LN, low N; NN, normal N.
Fig.4.Effects of BRs on N accumulation under different N conditions.(a)N accumulation in shoots and roots of maize seedlings supplemented or not with eBL at 6 days after LN or NN supply.(b)N accumulation in shoots and roots of wild-type and zmbri1 plants at 6 days after LN or NN supply.The control and WT are maize cv.B73.Values with error bar are mean±SD(n=3).Different letters indicate significant difference between treatments in shoots and roots by Duncan’s multiple-range test at P<0.05.eBL,2,4-epibrassinolide; WT, wild type; LN, low N; NN, normal N.
Root architecture plasticity is crucial for many plant species suffering from soil nutrient limitations, and N supply affects root growth and development inArabidopsis, rice, and maize [46-48].Studies [49,50] suggest that BRs influence the root growth and development of various plant species.BR-deficient or -signaling mutants displayed short-root phenotypes [1,2,51].Exogenous low concentrations of BRs increased root growth, whereas high concentrations of BRs repressed it [1,52].In the present study,exogenous application of eBL promoted primary root growth under both LN and NN conditions, whereaszmbri1plants showed shortroot phenotypes in contrast to wild-type plants.LN induced growth of primary roots in maize seedlings, and exogenous eBL promoted the effects of LN-mediated primary root elongation,whereas knockdown ofZmBRI1weakened the biological effects of LN-induced primary root elongation.In a previous study [8], LN up-regulated the transcript of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation, whereas a mutant ofbsk3in BR signaling effectively repressed low N-mediated primary root elongation.LN also up-regulated the expression of BR biosynthesis geneAtDWF1contributed to the root foraging response [9].In agreement with these results, the present study suggested that BRs were involved in-modulated root architecture in maize seedlings.Further research could reveal the physiological and molecular mechanism by which BRs influence-modulatedroot elongation.
Fig.5.Changes in plant biomass and N accumulation in wild-type and zmbri1 plants under LN and NN conditions.(a,b)Dynamics changes in shoot(a)and root(b)dry weight of wild-type and zmbri1 plants at 1,3,6,and 9 days after LN or NN supply.Vertical bars indicate LSD at 0.05 levels.(c,d)Relative inhibition rate in shoot(c)and root(d)dry weight of wild-type and zmbri1 plants.Relative inhibition rate = (NN - LN)/NN.(e, f) Dynamic changes in N accumulation in shoot (e) and root (f) of wild-type and zmbri1 plants at 1,3,6,and 9 days after LN or NN supply.Vertical bars indicate LSD at 0.05 levels.(g,h).Relative inhibition rate of N accumulation in shoot(g)and root(h)of wildtype and zmbri1 plants.Values with error bars are mean±SD(n=3).Different letters indicate significant difference between different treatments at the same time point by Duncan’s multiple-range test at P < 0.05.WT, wild type; LN, low N; NN, normal N.
Fig.6.Knockdown of ZmBRI1 influenced uptake.(a,b)Net fluxes(a)and mean fluxes(b)in roots of wild-type and zmbri1 plants under LN and NN conditions.Values with error bars are mean±SD(n=8).(c,d) 15N content(c)and 15influxes(d)after 10 min 15N tracing assay in wild-type and zmbri1 roots.Values with error bars are mean±SD(n=3).Different letters indicate significant difference between treatments by Duncan’s multiple range test at P<0.05.WT,wild type;LN,low N;NN,normal N.
BRs function in the regulation of physiological and developmental processes in plants and also modulate N uptake and metabolism in plants [13,53].In the present study, exogenous application of eBL increased N accumulation in shoots and roots of maize seedlings exposed to LN or NN treatment.Correspondingly,zmbri1plants showed lower N accumulation in shoots and roots than did wild-type plants under both LN and NN conditions.Knockdown ofZmBRI1altered the relative inhibition rate by LN of N accumulation.Although the lower growth rate of zmbri1plants led to low nutrient demand, the relative inhibition rate of dry biomass in shoots showed no significant difference between wild-type andzmbri1plants under the LN condition, while that inzmbri1plants showed a slight upward tendency in comparison with wild-type plants subjected to extended LN treatment.Thus, the higher inhibition rate of N accumulation inzmbri1plants appears not to be the major limiting factor in plant biomass accumulation during short-term N deficiency, and thezmbri1plant biomass inhibition rate may depend on the cumulative time under LN conditions.
Associated with their N accumulation,zmbri1plants showed lowerfluxes and15uptake than wild-type plants under both LN and NN conditions.Similar results were observed [15] in cucumber, where exogenous application of eBL promotedandflux rates.These findings indicate that BRs are involved in the process of N uptake for increasing N accumulation in maize seedlings.NRT protein families have been shown [20] to be involved inabsorption from soil and translocation to various plant tissues in many species.The expression of NRT genes is regulated by auxin, ethylene, cytokinin, and gibberellin in plants in response to N supply [30,54].Previous studies [15,53] indicated that BRs regulated the transcription of NPF/NTR1 genes inArabidopsisand cucumber.ZmNPF6.6is a dual-affinity nitrate transporter protein, whereasZmNPF6.4has low-affinitytransport activity [22].In the present study, the expressions ofZmNPF6.4andZmNPF6.6were down-regulated inzmbri1roots in comparison with wild-type roots under LN and NN conditions.In contrast,eBL treatment increased the transcript expression ofZmNPF6.4andZmNPF6.6relative to the control under both LN and NN conditions.AtNRT2.1is the major high-affinity transport gene responsible foruptake at low concentrations ofinArabidopsis[17].The expression levels ofZmNRT2.1andZmNRT2.2,two maize homologs ofAtNRT2.1were also correlated withuptake capacity in response to N supply in maize [21].In the present study,zmbri1roots showed lower expression levels ofZmNRT2.1andZmNRT2.2than wild-type roots under both LN and NN conditions.But eBL treatment increased the transcript expression ofZmNRT2.1andZmNRT2.2relative to the control.Thus,zmbri1roots showed lower transcription levels of NRT genes, leading to lowerfluxes and15uptake.These findings indicate that BRs could modulate the transcript expression of NRT genes to alter N uptake in maize in response to N supply.The regulatory interactions between BRs and NRT genes invite further study.
Fig.7.Changes in transcript expression of ZmNPF6.4, ZmNPF6.6, ZmNRT2.1, and ZmNRT2.2 in zmbri1 (a-d) and eBL-treated (e-h) roots under LN and NN conditions.The control and WT are maize cv.B73.Values with error bars are mean±SD(n=3).Different letters indicate significant difference between treatments at the same time point by Duncan’s multiple range teste at P < 0.05.eBL, 2,4-epibrassinolide; WT, wild type; LN, low N; NN, normal N.
N supply influenced the transcript expression of genes involved in BR biosynthesis and signal transduction, and exogenous eBL application increased root length,plant biomass,and N accumulation in maize seedlings.zmbri1plants showed shorter roots and less plant biomass and N accumulation than wild-type plants under both LN and NN conditions.zmbri1plants showed lower netfluxes and15uptake than wild-type plants.The transcript expression ofZmNRTgenes was down-regulated inzmbri1roots relative to that in wild-type roots after N treatments.Exogenous application of eBL up-regulated the expression ofZmNRTgenes.Thus,BRs modulate N physiological response and regulate the transcript expression of NRT genes to promote N uptake in maize,suggesting a way to improve N efficiency in maize production.
CRediT authorship contribution statement
Jiapeng Xing:designed the research, performed experiments,and drafted the manuscript.Yubin Wang, Qingqing Yao, and Yushi Zhang:performed part of the work.Zhaohu Li:provided technical assistance.Mingcai Zhang:conceived the project and revised the manuscript.
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 research was supported by National Key Research and Development Program of China (2017YFD0300410).We thank Dr.Philip W.Becraft and Dr.Yanhai Yin (Genetics, Development, and Cell Biology Department of Iowa State University,USA)for supplying theZmBRI1RNA interference (RNAi) mutants and excellent technical assistance.
Appendix A.Supplementary data
Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.04.004.