Goyun Song,Guoling Sun,Xingchen Kong,Meiling Ji,Ke Wng,Xingguo Ye,Yun Zhou,Shuifeng Geng,Long Mo,*,Aili Li,*

aNational Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

bCollaborative Innovation Center of Crop Stress Biology,Institute of Plant Stress Biology,School of Life Science,Henan University,Kaifeng 475004,Henan,China

Keywords:Floret development Spike morphology Sterile lemma Wheat

ABSTRACT The Q gene in common wheat encodes an APETALA2(AP2)transcription factor that causes the free threshing attribute.Wheat spikelets bearing several florets are subtended by a pair of soft glumes that allow free liberation of seeds.In wild species,the glumes are tough and rigid,making threshing difficult.However,the nature of these “soft glumes”,caused by the domestication allele Q is not clear.Here,we found that over expression of Qincommon wheat leads to homeotic florets at glume positions.We provide phenotypic,microscopy,and marker genes evidence to demonstrate that the soft glumes of common wheat are in fact lemma-like organs,or so-called sterile-lemmas.By comparing the structures subtending spikelets in wheat and other crops such as rice and maize,we found that AP2 genes may play conserved functions in grasses by manipulating vestigial structures,suchasfloret-derived soft glumesin wheat and empty glumes in rice.Conversion of these seemingly vegetative organs to reproductive organs may be useful in yield improvement of crop species.

1.Introduction

The free-threshing trait in common wheat permits mechanical harvesting of this important staple crop.This trait is largely conferred by the well-known domestication gene Q,which has pleiotropic functions on shaping the modern wheat cultivars including shorter,square-headed spikes,free-threshing grains,soft glumes,tough rachis and earlier heading[1-8].

Q is a member of the APETALA2(AP2)family of transcription factor genes[9].This family is named after the Arabidopsis AP2 gene that is characterized by an AP2 DNA binding domain that is unique to plants[10].Arabidopsis AP2 is a primary member of class-A genes that specify whorl 1 and 2 organ identity[10,11].Homologs of the wheat Q gene include the maize indeterminant spikelet1(ids1)gene[12]and OsIDS1 gene in rice[13].Although the regulatory targets of AP2 genes appear to be conserved between monocots and eudicots[14,15],the individual genes have adopted novel functions in monocots.The diverged functions of orthologous AP2 genes may account for different inflorescence structures in different species.For example,the maize indeterminate spikelet1(ids1)and related sister of ids1(sid1)genes are needed for branching of inflorescence meristems to initiate floral meristems and to control spikelet meristem determinacy[16].In rice,SUPERNUMERARY BRACT(SNB)and OsIDS1 are involved in lodicule development.In maize,spikelets of ids1 sid1 double mutants produce continuous glume-like structures without floral organs[16].Rice snb osids1 double mutants,however,generate numerous glume-like structures in the spikelets, although they eventually produce floral organs [13]. Qmutants produce longer spikes and thicker and shorter glumes,but show no change in floret and glume numbers.Compared with wild type wheat spikes Q mutants have harder glumes and are more difficult to thresh[17].The anatomical and molecular nature of such changes remains illusive.

Here,we show that overexpression of the Q gene reduces the length of spike and increases the number of florets.More interestingly,the glumes appear to be homeotically changed into lemma-like organs or even fertile florets.We performed a detailed structural comparison of wheat glumes and lemmas and found that they were anatomically similar.This was supported by expression patterns of lemma marker genes.We propose that the soft glumes of common wheat have adopted a lemma identity.

2.Materials and methods

The spring type wheat cultivar CB037 was used to produce transgenic plants because of its short life cycle and ease of transformation.Plants were grown in a greenhouse at 22°C day/20°C night,16 h light/8 h darkness,and 50%relative humidity.The full-lengthc DNA of Q was cloned into the vector pCAMBIA3300 with the maize ubiquitin promoter and was transformed into CB037 by an Agrobacterium-mediated approach.Floral organs or tissues were collected from wheat plants at the booting stage,frozen in liquid nitrogen,and stored at-80°C until RNA extraction.All tissues were collected with two biological replicates.Total RNA was extracted using the TRIzol reagent(Invitrogen)and ～1 μg of RNA was used for reverse transcription and subsequent real-time PCR(Perfect Real Time,TaKaRa).Gene expression levels were normalized relative to the wheat actin gene[17].For scanning electron microscopy(SEM),young spikes and sectioned glumes were fixed in 2%glutaraldehyde in 0.2 mol L-1phosphate buffer(pH 7.2),and were then dehydrated in astandard ethanol series and dried usingCO2.The tissues were then coated with platinum.A scanning electron microscope(SEM SU8010,Hitachi,Ltd.)was used for observation and photography.

3.Results

3.1.Overexpression of Q converts glumes into florets or floretlike organs in common wheat

The Q allele,which confers wheat domestication traits,is more abundantly transcribed than the primitive q allele.Moreover,the two alleles are distinguished by a single valine to isoleucine(Val329Ile)conversion that may enhance homodimer formation during protein-protein interactions[9].Mutations at the miR172 binding site, including this conversion in the Qgene,were found to be sufficient to increase transcript levels by reducing miRNA-dependent degradation.The moderate increase in Q expression level enhanced the function of the Q allele[18,19].Here,we studied the effect of Q in overexpression line sand found that up to 50%of spikelets had lost one or both outer glumes due to homeotic conversion to floret-like structures.Partially developed florets also lacked organs such as stamens or a palea(Fig.1-A-D,F),but fully developed lateral florets(LFs)were fertile and produced grains,resulting in an increased number of grains per spike(Fig.1-E-F).Moreover,many glumes on spikelets of Q overexpression lines were observed to carry awns that are usually present only on lemmas and these awns were longer than those on the wild type genotype(Fig.1-G-H).The development of these awns that was comparable to those on fully developed LFs and wild type florets (Fig. 1-I-J) suggests that glumes in lines overexpressing Q had converted into lemma-like organs(Llos). Observation of these Llos under SEM revealed that some glume primordia were indeed replaced either by lemmalike primordia(Fig.1-K-L),or by floral primordia(Fig.1-M-N).These observations indicate that SGs had taken the identity of lemmas(Les)in common wheat.

Fig.1-Characterization of the soft glumes in common wheat.The phenotypes of a wild type spikelet(A)and spikelets from overexpression Line#3(Q3)with varying portions of soft glumes being homeotically converted into florets with 2 stamens in the left floret and 1 stamen in the right floret(B),with 1 stamen on the left floret and 3 on the right one(C),and with one pistil in the left floret and 3 stamens in the right(D).Lemmas and paleas were removed to expose stamens.*indicates positions of stamens.E and F represent three spikelets of the wild type (E) and three of LineQ3 (F).Awnswere removed for better display of Llos or florets with seeds in (F). G-J show awn development in SG (G), Llo (H) and lemmas of the wild type (I), and Line Q3 (J) taken from the bottom to the top of a spike.K-L,SEM images of the top portion of an inflorescence at the anther meristem(AM)stage from the wild type(K)and Line Q3 showing that soft glume primordia in(K)were converted to lemma-like organ primordia in(L).M and N are enlarged SEM images of the middle sections of inflorescences showing aglume with one floret(M)and two florets with one converted from the glume(N).AM indicates positions of stamen and pistil meristems.O and P,cross sections of glumes(O)and Llo(P)from the wild type and Line Q3 respectively.M and N are lemmas from the same corresponding plants.S-V are squared regions in O-R with larger amplification showing similar arrangements of sclerenchymatous cells at the abaxial side and parenchymal cells at the adaxial side of corresponding organs.W,relative expression levels of the Q gene in the wild type and over expression L ines Q11,Q2,and Q3.X and Y,expression levels of lemma identity genes in SGs and Llos(X)and corresponding lemmas(Y)respectively.Z,relative expression of lemma marker genes in tough glumes of Ae.tauschii and its lemmas.M1,M6,M34,and DL represent wheat homologs from the rice MADS1,MADS6,MADS34 and DROOPING LEAF genes,respectively.SG,soft glume;TG,tough glume;Llo,lemma-like organ;Le,lemma;GP,glume primordium;LP,lemma primordium;FM,floret meristem;AM,anther meristem;PC,parenchymal cells;SC,sclerenchymatous cells.Scale bars,A-J and O-R,1 cm,K-L,50 μm,M-N,200 μm,S-V,100 μm.

3.2.The soft glumes of common wheat are actually lemma-like organs

To further characterize the structural nature of SGs,Llos,and Les,we dissected them vertically and studied their cell compositions under SEM.As shown in Fig.1-O-V,SGs,Llos,and Les had similar cell structures,with sclerenchymatous cells at the abaxial side and parenchymal cells at the adaxial side,suggesting that these organs have common identity.

We further characterized these organs using marker genes.In rice,the SEPALLATA-like gene OsMADS34 is involved in rudimentary lemma identity,and its protein forms protein complexes with two other MADS-box proteins,OsMADS6 and OsMADS1[20-24].These MADS-box genes,together with DROOPING LEAF(DL),which is regulated by OsMADS34,are considered marker genes for lemma identity in rice[22].We identified the best homologs of these rice lemma marker genes in wheat and studied their expression patterns in soft glumes and related organs[25].We firstly showed that in the three Q overexpression lines(OEQL11,OEQL2,and OEQL3),the Q gene expression level was increased 14-,26-,and 37-fold compared to the wild type(Fig.1-W).We then showed that all four lemmamarker gene homologs displayed similar expression patterns in SGs,Llos,and Les(Fig.1-X-Y).In contrast,these genes showed significantly different expression levels between glumes and lemmas in Aegilops tauschii,which has undomesticated tough glumes,especially in the case of TaDL(Fig.1-Z).These data further support the notion that soft glumes in common wheat are lemma-like organs or sterile lemmas.

4.Discussion

The free threshing trait makes it much easier to harvest wheat grains.This domestication step involved a subtle sequence change in q that led to Q.Despite sequence similarity,orthologs of AP2 genes diverged to perform different functions in different species.In maize,for instance,double mutants of ids1 and sid1 resulted in indeterminate spikelet meristem and reiterated development of lemma-like bracts with no floret meristems[12,16].In rice,the mutations of OsSNB produced extra rudimentary glumes[26],while the mutation of MULTI-FLORET SPIKELET1(OsMFS),another AP2 gene,caused ectopic development of extra hull-like organs(lemma or palea like organs)in the spikelet[27].These studies indicate that the Q gene and its related sequence orthologs play diverse roles in modifying spikelet composition,and hence inflorescence structures,in various grass species.

The common wheat spike is composed of various numbers of spikelets,which are subtended by a pair of soft glumes at the base with 3-6 florets in the middle.The Q/q alleles have dosage effects on spike morphology,and both are regulated by miR172[5,11].In fact,Q is incompletely dominant to q[9].Five copies of q cause square-headed spikes in Chinese Spring[5].Mutation at the miR172 binding-site in 5Dq caused spike compactness and reduced plant height[28].However,little is known about the function of Qinglume development;mutation of Q in common wheat converts soft glumes to tough ones like those in Ae.tauschii,indicating that Q has a role in glume development[17].Likewise,mutationat themiR172 bindingsite of the Q allele also causes soft glume deficiency[18,19].Here we show that overexpression of Q caused homeotic conversion of soft glumes into florets or floral organs.Morphological and microscopy experiments showed that the soft glumes lost their real-glume features.Expression patterns of lemma marker genes indicated homogeneity of soft glumes and lemmas,which displayed different expression patterns between(tough)glumes and lemmas in Ae.tauschii,the D genome donor of common wheat.Here we show that increasing the expression level of Q can improve the floret numbers in common wheat through conversion of soft glumes into florets.Another gene,Tenacious glume(Tg),is also involved in glume development,but its role is still unclear[29].

Plants modify their inflorescence structures to adapt to the environment,or as a response to human selection.In rice,“empty glumes”on spikelets have been shown to be sterile lemmas or rudimentary lemmas[22].On the other hand,ectopic expression of LATERAL FLORETS(LF1)causes the initiation of lateral meristems to generate lateral florets at the axil of the sterile lemma[30].Further comprehensive comparison may reveal a unified molecular network to regulate grass inflorescence development.Such knowledge may be useful in better understanding yield components,such as grain number per spike,in crops and assist in their improvement.

Acknowledgments

We are grateful for Dr.Justin Fairs for critical reading of this manuscript.This work is supported by the National Key Program for Transgenic Crop Cultivation(2016ZX09001-001)and The CAAS Agricultural Science and Technology Innovation Program Cooperation and Innovation Mission(CAAS-XTCX2016).