Shripad Ramchandra Bhat ,b,3 and Yelam Sreenivasulua,3,4
aBiotechnology Division, Council of Scientific and Industrial Research CSIR-Institute of Himalayan BioresourceTechnology, Palampur 176061, Himachal Pradesh, IndiabIndian Council of Agricultural Research ICAR-National Research Centre on Plant Biotechnology, New Delhi110012, India
In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals,these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstreameffector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led usto investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant andresulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, alteredMegaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features thatoverlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant allelespointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expressionof various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis andtransport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant andthe altered expression of key genes related to gametophyte and seed development could be explained based on the functionalsimilarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediatedproteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.
The life cycle of higher plants comprises a short,haploid, gametophyte phase and a long, diploid, mul-ticellular, sporophyte phase. The transition to the
gametophyte phase is initiated when a diploid sporemother cell differentiates and undergoesmeiosis to giverise to four haploidmegaspores. In the ovule, of the fourspores, the three that are closest to the micropylar endof the ovule degenerate while the fourth, closest to thechalazal end, becomes a functional megaspore anddifferentiates into a megagametophyte after typicallythree cycles of free nuclear mitotic divisions followedby highly polarized cellularization (Yang et al., 2010;Sprunck and Gross-Hardt, 2011). The resulting embryosac is the mature female gametophyte (FG). A typicalFG comprises a seven-celled embryo sac containing sixhaploid cells (an egg cell, two synergids, and three an-tipodals) with one diploid central cell. Likewise, fol-lowing meiosis, the male microspore undergoes anasymmetric cell division resulting in a male gameto-phyte comprising one vegetative cell and two spermcells (Twell, 2011).
The haploid gametes develop in the midst of diploidmaternal tissue, which requires constant communicationbetween the two types of cells. After fertilization, inter-communications among the embryo, the endosperm,and the seed coat, each with a unique genetic identity, isessential for proper seed development (Figueiredo andKöhler, 2016). During the alternation of generations, cells
1 This work was funded by the Council of Scientific and IndustrialResearch (project nos. MLP-072 and BSC-0107), New Delhi, India,and the Indian Council of Agricultural Research, New Delhi, India,through the National Agricultural Innovation Project (NAIP-4157).
2 Deceased3 Address correspondence to [email protected] and sreenivasulu@
ccmb.res.in.4 Current address: Council of Scientific and Industrial Research-
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad500007, India.
The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Yelam Sreenivasulu ([email protected]).
Y.S. conceived the original screening and research plans and su-pervised the experiments; S.K.S. performed most of the experiments;V.K. performed microarray experiments and qRT-PCR analysis.; R.S.,P.S.A., S.R.B., and Y.S. designed the experiments and analyzed thedata; S.R.B. and Y.S. conceived the project and wrote the article withcontributions of all the authors; Y.S. supervised and complementedthe writing.
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have to undergo two transition phases: the first transitionoccurs during the specification of gametophytic cellsfrom sporophytic cells (archesporial cell formation),which is designated as a mitotic-to-meiotic transition,and the second occurs during the development of thegametophyte, which is designated as a meiotic-to-mitotic transition (Van Durme and Nowack, 2016). Anumber of mutations affecting various stages of malegametophyte or FG development have been identifiedand reviewed (Liu and Qu, 2008), and the role ofphytohormones and lipid molecules in controllingthese developmental stages through complex signal-ing has been documented (Nakamura, 2015; Schusteret al., 2015). Cloning and characterization of suchgenes (Liu and Qu, 2008; Drews and Koltunow, 2011)have provided valuable insights into the molecularevents underlying these critical stages in the life cycleof a plant. However, the molecular mechanisms thatcoordinate the germline, zygote, and seed develop-ment in plants are as yet only poorly understood(Jurani�c et al., 2012).Convincing evidence now available with animal cells
suggests that the transition and progression of the cellcycle and cell fate determination are temporally andspatially controlled by targeting key regulators throughthe ubiquitin/26S proteasome pathway (McCarthyCampbell et al., 2009). Studies on mammalian cells andCaenorhabditis elegans have revealed that multisubunitCullin3 (CUL-3)-RING E3 ligases (CRL) regulate dif-ferent cellular and signaling processes during themeiosis-to-mitosis transition (Bowerman and Kurz,2006; Sawin and Tran, 2006; Sumara et al., 2008). Rapiddegradation of meiotic proteins is a prerequisite for theswitch to mitotic division (the oocyte-to-embryo tran-sition) in nematodes. The earliest mitotic spindle differsfrom its meiotic counterpart in that the latter is labeledwith meiosis-specific proteins that are earmarked fordegradation so as to avoid any interference from themeiotic spindle during subsequent mitotic divisions(Lu and Mains, 2007). A female germline-specific CUL-3substrate adaptor, namely the Maternal Effect Lethal26(MEL-26) consisting of aMATH (Meprin-Associated TrafHomology) domain and a Broad-complex, Tramtrack,Bric-a-brac (BTB) domain, is responsible for the spatialand temporal targeting and degradation of MEI-1/katanin (Defective in Meiosis1) during the meiosis-to-mitosis transition to allow the cells to enter the mitoticstage. Similarly, MEL-26 targets the microtubule-interacting protein Fidgetin-Like1 for degradation dur-ing mitosis (Luke-Glaser et al., 2007). Weber et al. (2005)showed that a MATH-BTB protein functions in a similarfashion in Arabidopsis (Arabidopsis thaliana), and Jurani�cet al. (2012) characterized similar proteins in maize (Zeamays).Cosson et al. (2010) identified 71 proteins in Arabi-
dopsis that contain the MATH domain and classifiedthem into four families. Of these, two belong to theubiquitin-specific protease 7 family, six to theMATHd/BTB family, and one to the MATHd/filament proteinfamily. The remaining 62 proteins, which possess up to
four MATH domains without any other associateddomains, belong to the MATHd-only protein familyand are classified as Tumor Necrosis Factor Receptor(TNF-R) Associated Factors like (TRAF-like) genes.TRAF proteins are widely found in metazoans andserve as adapter proteins that help in the transmissionof external signals received by the TNF-R to down-stream effector molecules. TRAFs are E-3 ubiquitin li-gase proteins that mediate the interactions betweenTRAF members and receptors and also those betweenTRAF members and several intracellular signalingmolecules (Zapata, 2003; Alvarez et al., 2010) by virtueof their MATH domain. In animals, TRAF proteinsregulate diverse cell processes, including immune re-sponse, inflammatory response, apoptosis, cancer,embryogenesis, and the survival of the cell itself(Kedinger et al., 2005). In plants, TRAF-like genes havebeen reported to play a role in pathogenesis (RTM3;Cosson et al., 2010), in abscisic acid-mediated droughtstress signaling (SINA2; Bao et al., 2014), and in insectherbivory (At5g26260 and At3g28220; Schweizer et al.,2013). However, no role has been reported so far in re-productive development; to our knowledge, this is thefirst such report. We found that a mutation in the TRAF-like gene (At5g26290) affected normal reproductive de-velopment in Arabidopsis. The At5g26290 gene isexpressed specifically during the development of themale and female germline in gametophytic and thesurrounding sporophytic tissue. Detailed expressionprofiling of this gene revealed its role inmodulating keygenes at various stages of gametogenesis and duringthe progression of embryo sac differentiation. There-fore, we have named the At5g26290 gene as the TRAFMediated Gametogenesis Progression (TRAMGaP) gene.
RESULTS
Mutation in TRAMGaP Causes Seed Sterility
While screening a T-DNA promoter trap populationof Arabidopsis generated in house, we noticed amutantwith short siliques and high (;50%) seed sterility (Fig.1; Table I). Using the genome-walking approach, theT-DNA insertion site was identified in the mutant(Supplemental Fig. S1A). A BLAST search of the se-quence amplified in the genome walk against the Ara-bidopsis genome database showed a complete matchwith the last exon of the At5g26290 gene between thecoordinates 9,227,747 and 9,228,082 of the fifth chro-mosome of Arabidopsis (Fig. 1E; Supplemental Fig.S1B), indicating that insertion of T-DNA had disruptedthe At5g26290 gene. This gene is known to code for aTRAF-like protein. The site of insertion of the T-DNAwas further confirmed by PCR using forward and re-verse primers specific to the T-DNA flanking region(Supplemental Fig. S1C). In kanamycin-positive T2progeny of this mutant, two amplicons were detected: a1.8-kb amplicon corresponding to the wild-type alleleand an ;7-kb amplicon coming from the mutant allele(Supplemental Fig. S1C). Sequencing of the ;7-kb
amplicon revealed the presence of two tandem copies ofT-DNA. Thus, PCR not only confirmed the insertion ofT-DNA into the At5g26290 locus but also helped toseparate plants that were homozygous for the mutantfrom those that were heterozygous for it.
For more detailed characterization of the At5g26290gene, another mutant line (SALK_0146328; Fig. 1C)with the site of T-DNA insertion in the fourth intronwas procured from theArabidopsis Biological ResourceCenter and the insertion site was confirmed as above.Of the two allelic variants, the mutant developed inhouse was designated as tramgap1-1 and the SALKmutant was designated as tramgap1-2 (Fig. 1E). Unlessstated otherwise, all statements in this article pertainingto the mutants refer to homozygous mutant plants.
A gene complementation study was undertaken toconfirm that the failure to set seed in tramgap1-1 was dueto loss of theTRAMGaP gene. Plantswith tramgap1-1weretransformed with the gene cassette 35S::TRAMGaP. Thetransgenic plants selected on hygromycin set seeds nor-mally, confirming the functional complementation of themutant phenotype by the transgene (Fig. 1D).
At5g26290 Is a Member of the TRAF-Like Gene Family
At5g26290 (TRAMGaP) is 1.75 kb long, containsseven exons, and is capable of coding for a polypeptideconsisting of 322 amino acids (Supplemental Fig. S2).Phylogenetic analysis of TRAF proteins of both the
Figure 1. TRAMGaPmutant phenotypes and location of the T-DNA insertion in themutants. A, Dissected silique from awild-typeplant showing well-filled, viable seeds. B, Homozygous tramgap1-1 silique at the stage comparable to that in the wild typeshowing normal and aborted ovules (asterisks). C, Homozygous tramgap1-2 silique at the stage comparable to that in the wildtype bearing normal and aborted ovules (asterisks). D, Silique from a genetically complemented homozygous tramgap1-1mutantplant showing full seed set. E, T-DNA insertion in tramgap1-1 and tramgap1-2 mutant lines. Bars = 500 mm.
Animalia and Plantae kingdoms, including human,mouse,Drosophila spp., Populus spp., rice (Oryza sativa),and Arabidopsis, using MEGA6 software (Tamuraet al., 2013) showed that the sequences were highlyconserved over long evolutionary periods (SupplementalFig. S3). However, proteins from the two kingdoms fellinto separate groups, indicating a clear divergence be-tween the two. In the phylogenetic tree, five closely re-lated Arabidopsis TRAFs fall into a single clade(At5g26260, At5g26280, At5g26290, At5g26300, andAt5g26320). Cosson et al. (2010) grouped all 69 MATHdomain-containing proteins of Arabidopsis into fourfamilies. Of these, 62 proteins, including At5g26290,have been assigned to the MATHd-only protein family,which contains up to four MATH domains without anyother associated domains. Most of the genes of thisfamily are organized into clusters (Cosson et al., 2010).At5g26290 belongs to the third largest cluster with fourother genes arranged in tandem (At5g26260,At5g26280,At5g26300, and At5g26320; Supplemental Fig. S4), is64% to 71% identical to At5g26290 at the level of aminoacids (Supplemental Fig. S5), and grouped separatelyfrom all other members of the family (SupplementalFig. S4). Thus, homology analysis clearly showed thatAt5g26290 is a TRAF-like protein.Going by TAIR annotation, At5g26290 is denoted as a
MATH domain-containing TRAF-like protein. Struc-turally, TRAF proteins share a conserved region ofabout 180 residues with meprins. At5g26290 containstwo MATH domains, and the Simple Modular Archi-tecture Research Tool (http://smart.embl-heidelberg.de) predicted two MATH domains at amino acid po-sitions 57 to 162 and 203 to 303 of the polypeptideencoded by At5g26290 (Supplemental Fig. S2). TheMATH domains contain eight b-strands, as shownin the secondary structure prediction analysis(Combet et al., 2000), that also are found in At5g26290(Supplemental Fig. S6). Quaternary structure analysisof TRAMGaP also revealed that one of the MATH do-mains was nearly identical (99.86%) to a TRAF-likeprotein, whereas the other MATH domain was nearlyidentical (99.94%) to a speckle-type POZ protein (SPOP;Supplemental Fig. S7). The alignment of At5g26290MATH domains with TRAFs reported from humansamples and of RTM3, a MATH protein reported inArabidopsis by Cosson et al. (2010), showed conser-vation at the level of amino acids for all eight TRAF-2b-strands (Fig. 2). In tramgap1-1, T-DNA insertion
disrupted the C-terminal MATH domain, whereas intramgap1-2, it was the N-terminal MATH domain thatwas disrupted (Supplemental Fig. S8).
TRAMGaP Is Expressed Mainly in Seedlings andReproductive Organs
Reverse transcription-PCR analysis showed TRAMGaPtranscripts in roots and aerial parts of 7-d-old seedlingsand in the inflorescence (Supplemental Fig. S9A). Quan-titative expression analysis using quantitative reversetranscription (qRT)-PCR also revealed the highest levelsof expression of this gene in 7-d-old seedlings and inflower buds (stages 5–9). TRAMGaP transcript levelsdecreased 3- to 7-log fold in various tissues such asstem, leaf, axillary branches, and cauline leaves dur-ing different floral developmental stages, namelystages 10 to 12 and 13 to 14, and were undetectableafter fertilization (stages 15–18; Supplemental TableS1). These results indicate that TRAMGaP is expressedspecifically in seedlings and in floral tissues, resultsthat are in agreement with the expression pattern ofTRAMGaP reported in the Genevestigator microarraydatabase (www.genevestigator.ethz.ch; SupplementalFig. S9B).
To further understand the tissue-specific expressionpattern of TRAMGaP, a 1.5-kb fragment upstream ofthe TRAMGaP coding sequence was amplified andused to assemble a pTRAMGaP::uidA construct. T2transgenic Arabidopsis plants carrying this gene cas-sette showed strong GUS expression in both micro-gametophytes and megagametophytes (Fig. 3). GUSexpression in ovules was found in nucellar tissue aswell as in all cells of the embryo sac (Fig. 3, A–E),whereas in anthers, GUS expression was localized tomicrospores or pollen and to tapetal cells (Fig. 3, F–J).Thus, TRAMGaP is expressed mainly in sporophyticand gametophytic tissues of reproductive structuresand within flowers, and the expression is limited to thenucellus and the tapetum.
Mutation in TRAMGaP Causes Seed Sterility, and MutantAlleles Are Transmitted through Both Male andFemale Gametes
Both the allelic variants of the tramgapmutanthad shortersiliques and fewer ovules in each silique compared with
Table I. Silique characteristics of tramgap mutant lines of Arabidopsis
Genotype Mean Silique Length Mean No. of Ovules in Each Silique
those of wild-type plants (Table I). Plants heterozy-gous for the mutation also had significantly fewer(;31) ovules in each silique and showed ;50% seedset (Table II). In plants homozygous for the mutation,seed sterility increased to 65% to 70% and the numberof ovules in each silique decreased further to 24 to28 (Table I; Fig. 1). In general, phenotypes of tramgap1-2 plants were affected more severely than those oftramgap1-1. Seed sterility ranging from 34% to 37%also was recorded in the crosses between wild-typeplants and the heterozygous (tramgap/+) mutantplants (Table II). Seed sterility in wild-type plantscrossed with pollen from heterozygous mutant plantssuggests that heterozygosity results in seed abortion.However, the recovery of homozygous mutant plantsupon selfing shows that the tramgap mutation is notcompletely lethal. These results indicate that hap-loinsufficiency of TRAMGaP in heterozygotes leads toseed sterility. In order to better understand in greaterdetail the transmission efficiency of the mutant allelesthrough male and female gametes, progeny of the re-ciprocal crosses between the wild type and tramgap1-1/+ were screened for kanamycin resistance, which islinked to the mutant allele. Nearly one-half of theprogeny of tramgap1-1/+ (♀) 3 wild type (♂) waskanamycin positive (Table III), indicating very highfemale transmission efficiency of the mutant allele. On
the other hand, the male transmission efficiency wasvery low (16.7%). Nearly normal female transmissionof the mutant allele and 37% seed sterility in the crossbetween heterozygous mutants and the wild type(with pollen from the wild type) suggest that themutation affects wild-type female gametes in hetero-zygous mutant plants through some sporophytic ef-fect. In contrast, low male transmission frequency andcomparable seed sterility in reciprocal crosses indicateboth sporophytic and gametophytic effects of themutation on the male side.
tramgap Mutants Display a Wide Range of Abnormalitiesduring Gametophyte Development by Hindering theSpecification of MMC and the Progressionof Gametogenesis
To understand the role of TRAMGaP in gametophytedevelopment,we compared the development of the ovuleand of the anther in the wild type with that in the twomutants. In the wild type, most of the premeiotic ovulesshowed only one MMC (Fig. 4A), and although a few(5.8%) showedmore than oneMMC (Fig. 4B), only one ofthem differentiated into a functional embryo sac. In con-trast, in tramgap mutants, 40% of the ovule primordiashowed several abnormally enlarged subepidermal cells
Figure 2. Amino acid sequence alignment of the MATH domain of TRAMGaP (At5g26290) with TRAF1, TRAF2, TRAF3, andTRAF5 of Human and RTM3 fromArabidopsis. The eight b-strands identified in TRAF proteins are indicated by horizontal arrows.Numbers in parentheses are amino acid positions corresponding to the start and end of eachMATH domain. Residues conservedbetween At5g26290 and at least one TRAF protein are shaded. At5g26290.1 and At5g26290.2 represent the first and secondMATH domains of TRAMGaP, respectively.
at the premeiotic stage (Fig. 4, C–E). Ovule develop-ment at anthesis also was examined to ascertain thesynchronization of ovule maturation with anthesis.Surprisingly, only 47.2% of ovules from the tramgap1-1/+ plants, 34.7% from tramgap1-1/tramgap1-1 plants,and 29% from tramgap1-2/tramgap1-2 plants hadattained maturity (stage FG7/8), whereas in wild-typeplants, the corresponding value was as high as 86.3%(Table IV; Fig. 4F). Approximately 31.3% of ovuleswere at the FG1 stage, displaying a teardrop-like cell(the functional megaspore) accompanied by threenuclei (Fig. 4J), a typical FG1 stage phenotype as de-scribed by Sundaresan and Alandete-Saez (2010). Insome ovules from tramgap1-1 and tramgap1-2, fournuclei arranged in a rowwere visible at the micropylar
end even at the FG4 stage (Fig. 4, H and I; SupplementalFig. S10, C and D). Such persistence of all four meioticproducts indicates impaired developmental programmedcell death (dPCD) during ovule development. In about8% of ovules, a degenerating embryo sac was observed,and about 44.8% of ovules lagged behind the FG7/8stage at the time of anthesis in the tramgap1-1/+ plants(Table IV): in tramgap1-1/tramgap1-1 and tramgap1-2/tramgap1-2 plants, such ovules accounted for 60.7% and63.3%of the total, respectively. Also, the differentiation ofthe chalazal megaspore into a functional megaspore wasaffected: instead of a typical seven-celled wild-type em-bryo sac (Fig. 4F), we found multinucleate megagame-tophytes with irregularly distributed nuclei (Fig. 4G). Intramgap1-1, embryo sacswith a variable number of nuclei
Figure 3. GUS expression in ovules (A–E) and anthers (F–J) of a transgenic (T1) Arabidopsis plant carrying the pTRAMGaP::uidAconstruct. A, GUS expression in the nucellus and MMCs in the premeiotic stage ovule. B, GUS expression in the functionalmegaspore and surrounding sporophytic tissue in the ovule at stage FG1. C to E, Ovule (stage FG6/7) showing GUS expression inthe gametophyte cells (synergids, egg cell, and central cell) at themicropylar end (C), at antipodals (D), and at the chalazal end (E).F, Anther from flower at stage 5 showing GUS expression in pollen mother cells. G, Anther from flower at stage 7 showing GUSexpression in tetrads and tapetal cells. H, Anther fromflower at stage 9 showingGUS expression in developingmicrospores and inthe tapetum. I, Anther from flower at stage 11 showing GUS expression in mature pollen and tapetum. J, Anther from flower at stage13 showing GUS expression restricted to mature pollen. AP, Antipodals; CC, central cell; EC, egg cell; S, synergid; T, tapetum.
Table II. Seed sterility in tramgap mutants from selfed plants and from various crosses
Cross (Female 3 Male) Normal Seeds Aborted Ovules or Seeds Sterility
(two to six nuclei) at the center also were observed,suggesting failure in the progression of mitosis after thecommitment of the functional megaspore (Fig. 4G). Thearrest of mitosis occurred at any of the three mitotic di-visions (Supplemental Fig. S10,A andB). Postfertilizationdefects alsowere evident in themutant: somedevelopingseeds contained only the endosperm (Supplemental Fig.S10F) or only the embryo (Supplemental Fig. S10G),whereas others had defective embryo and endosperm(Supplemental Fig. S10H). Such abnormalities could re-sult from defects related to fertilization by defectivepollen or of defective ovules.
To ascertain cell specifications in defective embryosacs, tramgap1-1 homozygous mutants were crossedwith lines expressing different cell-specific markers,and ovules from the F1 plants were examined for theexpression of the marker genes. The marker specific tosynergid cells (ET884) showed expression in two mi-cropylar cells of the embryo sac of wild-type ovules andin normal ovules of the mutant siliques (Fig. 4, K andM), but no GUS expression was observed in the embryosac of defective ovules (Fig. 4L). Likewise, the markerspecific to the central cell (DD65) also failed to showexpression in defective ovules (Fig. 4, P and Q). Theseresults indicate that the nuclei observed in the micro-pylar region of the embryo sacs from defective ovulesare either haploid megaspores, the development ofwhich is arrested after meiosis because of the loss ofdPCD, or mitotic products of megagametophytes, thedevelopment of which is arrested after two mitotic di-visions. Thus, the tramgap mutation affects gameto-phyte development by hindering the progression ofmitosis and cell specification in functional megaspores.
The tramgap mutants showed reduced pollen viabil-ity and low-frequency transmission of the mutant allelethrough the male line. Therefore, we investigated pol-len development in wild-type and mutant plants. Themicrospore mother cells underwent meiosis, giving riseto four haploid cells in wild-type and mutant plants(Fig. 5). However, morphological defects became visi-ble at the microspore stage corresponding to flowerdevelopment stage 9. At this stage, compared with thewild type, the microspore wall was not well formed inthe tramgap mutants (Fig. 5, A–C), and degeneration ofmicrospores became evident at flower developmentstage 11 (Fig. 5, D–F). In the wild type, the tapetumunderwent dPCD and pollen grains were fully devel-oped, with sporopollenin in their outer walls (Fig. 5D).In contrast, anthers in the mutant plants showed a mixof sterile, empty, and degenerating pollen grains and
normal mature pollen grains (Fig. 5, E and F). Fur-thermore, the tapetal cells were not fully degenerated,once again pointing to the loss of dPCD. Anther de-hiscence in the mutant was comparable to that in thewild type (Fig. 5, G–I) and occurred around flowerdevelopment stage 13. No other defects were observedin other cells of the anther, suggesting that the mutantphenotype in anthers is confined to themicrospores, thetapetum, or both. Staining mature anthers with Alex-ander’s stain showed that nearly 100% of pollen grainswere viable in the wild type (Fig. 6, A and C), whereasin the mutants, the proportion was only about 30% (Fig.6, B and D). Scanning electron microscopy showeduniform, elliptical pollen in the wild type (Fig. 6E),whereas defects in the pollen wall were observed inboth heterozygous and homozygous tramgap1-1 mu-tant plants (data not shown). Likewise, a mix of normaland irregularly shaped, shriveled pollen was seen intramgap1-1 mutants (Fig. 6F). Staining of mature pollenfrom the tramgap1-1/+ plants with 49,6-diamidino-phenylindole (DAPI) showed uninucleate, binucleate,and trinucleate pollen aswell as dead pollen that lackednuclei (Fig. 6H). In contrast, only trinucleate pollen wasobserved in the wild type (Fig. 6G; Table V). Both stainsshowed that approximately, 38.7% of pollen was ap-parently normal and trinucleate (Table V). Thus, thedefects observed in the embryo and the endosperm inthis mutant probably were due to the failure of doublefertilization: pollen carrying only a single sperm canfertilize either the central cell or the egg cell but notboth. As a result, further development of the seedwould be arrested owing to the lack of endosperm or ofembryo. In all cases of the failure to set seed, embryosnever progressed beyond the globular stage. Thus, eventhe pollen carrying the wild-type TRAMGaP alleleproduced on heterozygous mutant plants appear to besomewhat defective, which supports the inference thatthe mutation also has a sporophytic effect.
Mutation in TRAMGaP Alters the Expression of Its CloselyRelated Homologs
As shown earlier, in the 69-member TRAF family,At5g26290 is grouped with its adjacent four tandemlyarranged members and placed in a separate clade. Totest whether these members share any redundancywith TRAMGaP, the expression pattern of its close ho-mologs, namely At5g26260, At5g26280, At5g26300, andAt5g26320 (63%–70% homology at the level of amino
Table III. Transmission efficiency of the tramgap1-1 allele
Figure 4. Female gametogenesis in the tramgapmutant. A, Wild-type (WT) ovule primordium showing a proper single MMC atthe premeiotic stage. B, Wild-type ovule primordium showing two MMCs at the premeiotic stage. C, Homozygous tramgap1-1 ovule primordium showing two MMCs at the premeiotic stage. D, Homozygous tramgap1-1 ovule primordium showing sixMMCs at the premeiotic stage (white arrows). In one of them, nuclear division has been initiated (red arrow). E, Homozygoustramgap1-2 ovule primordium showing multiple abnormal MMCs at the premeiotic stage (white arrows). F, Wild-type ovule atanthesis (FG7/8) showing a fully differentiated proper embryo sac. G, Heterozygous tramgap1-1 ovule showing a multinucleatemegagametophyte with irregularly distributed nuclei (asterisks) at the center of the abnormal embryo sac. H, Homozygous
acids) was studied using reverse transcription-PCR.Transcripts of all four of these genes were less abun-dant than those of TRAMGaP in wild-type flower budsof stages 5 to 9 but were 3- to 5-log fold higher in flowerbuds of stages 10 to 12. At the postfertilization stage,siliques of wild-type plants contained transcripts ofAt5g26260, At5g26280, At5g26300, and At5g26320, butthose of TRAMGaP were undetectable (Table VI). qRT-PCR failed to detect the transcripts of TRAMGaP in thetramgap1-2mutant at any stage of flower development,nor was the expression of the At5g26300 gene detect-able in flower buds at stages 5 to 9 and 10 to 14, whereastranscripts of At5g26280 showed more than 2-log foldup-regulation at all the flower stages tested. Likewise,the expression of At5g26320 was up-regulated 3-logfold during flower stages 5 to 9. Thus, At5g26260,At5g26280, At5g26300, and At5g26320 showed over-lapping expression with TRAMGaP, especially duringflower stages 12 to 14, and loss of At5g26290 activitychanged the expression of other closely related homo-logs, suggesting some cross talk or redundancy amongthese genes.
Mutation in TRAMGaP Alters the Expression of Kinases,Transcription Factors, Proteases, and Genes Involved inLipid Biosynthesis
Considering that TRAFs are adaptor molecules,which assist signal transduction in different biologicalprocesses, the pleiotropic effect of the tramgapmutationis not unexpected. Microarray analysis to ascertainthe possible involvement of TRAMGaP in various de-velopmental processes showed a total of 3,150 dif-ferentially expressed genes in flower buds betweentramgap1-2 homozygous plants and the wild type. Ofthese, 1,707 genes were down-regulated and 1,443 wereup-regulated. We found differential regulation of132 kinases involved in developmental cell growth,protein autophosphorylation, and male gamete gener-ation and 19 genes involved in ubiquitination and 18 F-box proteins in the tramgap1-2 mutant (SupplementalTable S2). A number of transcription factors also werefound to be differentially expressed. These includedthose that contained the AP2 domain (10), those of thebHLH family (nine), zinc finger (55), CONSTANS-LIKE1 (four), MYB (16), those that contained the NACdomain (12), Arabidopsis Response Regulators (eight),
those of the PHD family (four), SCARECROW-LIKE(three), AGAMOUS-LIKE (four), BTB (three), bZIP(three), and 45 others. In addition, down-regulation wasobserved in 58 genes involved in lipid biosynthesis and in37 genes of the phenylpropanoid pathway. Furthermore,seven genes related to the metabolism of very-long-chainfatty acids, 15 genes related to lipid transfer proteins, sixgenes related to elongase b-ketoacyl-CoA synthase andfatty acid desaturases, and one, namely CYP86A1 (whichcatalyzes the v-hydroxylation of fatty acids involved inlipid biosynthesis), also were found to be differentiallyexpressed in the homozygous tramgap1-2mutants, alongwith a number of tapetal oleosin and APG-like genes.
The protein-protein interaction study confirmed thatall of these genes form a major cluster (comprising570 genes), indicating their probable function in dif-ferent molecular and biological processes leading toreproductive success. Ontological classification clearlyindicated that the differentially expressed genes areinvolved in regulating transcription, lipid biosynthesis,transport, hormone-mediated signaling, and gameto-phyte development (Supplemental Fig. S11). Pheno-typic as well as microarray analyses suggested that lossof TRAMGaP function affects some key genes related tothe transition phase of plant growth and development.
For further confirmation of differential expression,genes such as katanin, AGOs, DICER-like, and SGS,which are crucial for sporophyte-to-gametophyte tran-sition signaling, were examined using qRT-PCR. Theexpression of several key genes related to hormonalsignaling and of some well-characterized genes playingan important role in gametogenesis and in lipid bio-synthesis also was examined in homozygous tramgap1-2plants using qRT-PCR (Fig. 7). A total of 56 genes wereanalyzed to validate and extend the microarray results(Supplemental Table S3). The expression levels of thesegenes ranged from 2-fold up-regulation to 36-fold down-regulation at floral stages 5 to 9, whereas at later stages,the expression ranged from 4-fold up-regulation to9-fold down-regulation. This is consistent with the factthat TRAMGaP expression peaks at flower stages 5 to9 (Table VI), and the consequences of its loss would bemost evident around these stages. The changes observedat later stagesmight exert cascading downstream effects.The results of qRT-PCR were in agreement with thosefrom the microarray for most of the genes. For example,genes CS5, ACS5, MS2, DICER2, AGO9, BAM1, AN3,ERD1, VANGUARD, QRT2, GA2oX, AGAMOUS-like,
Figure 4. (Continued.)tramgap1-1 ovule showing four linearly arranged doughnut-shaped (arrows) nuclei at the micropylar end of the embryo sac. I,Homozygous tramgap1-2 ovule with four nuclei in a rectangular cluster in the micropylar region. J, Homozygous tramgap1-1 ovule arrested at stage FG1 showing a pear-shaped functional megaspore. K, Expression of a synergid cell marker (ET-884) in thewild type. L, Absence of expression of the synergid cell marker in the nuclei of the tramgap1-1 mutant embryo sac. M, Normalovule from a heterozygous mutant plant showing expression of a synergid cell marker. N, Differential interference contrast (DIC)image of a wild-type ovule carrying a central cell marker (DD65). O, Fluorescence image of the ovule in N showing expression ofa central cell marker (DD65). P, DIC image of a tramgap1-1 ovule carrying a central cell marker (DD65). Q, Fluorescence imageof the ovule in P lacking expression of the central cell marker (DD65). Defective cells/nuclei aremarkedwith asterisks and arrows.CC, Central cell; EC, egg cell; ES, endosperm; S, synergid cell.
MYB24, and Jasmonate were down-regulated at stages5 to 9 (preanthesis) in both microarray and qRT-PCRanalyses. The level of down-regulation was compara-ble for genes such as argonautes and katanin involved ingene repression mediated through the small interferingRNA (siRNA) pathway. The dynamics of argonautes areknown to be regulated by genes such as RDR2, RDR6,DCL3, SGS3, AGAMOUS, APETALA1, and LEAFY,which also were down-regulated in homozygous tram-gap1-2 plants (Supplemental Table S3).
TRAMGaP Regulates the Specification of MMC throughInteraction with AGO Proteins
qRT-PCR analysis showed down-regulation of argo-naute, dicer-like, and SGS genes, which play a crucialrole in RNA interference-mediated signaling to control
the differentiation of MMC progenitor cells. This obser-vation prompted us to examine whether the tramgapmutants also display phenotypes similar to those of theago9 mutation at the premeiotic stage. Indeed, out of135 ovules examined, about 40% displayed the ago9-likephenotype (i.e. the presence of multiple MMCs in thenucellar region; Fig. 4, C–E), whereas the remaining 60%of ovules showed the wild-type-like phenotype (Fig.4A). Postmeiotic ovules also showed ago9-like pheno-types (Fig. 8, A and B) in which abnormal gametic cellsas well as degenerating and functional megaspores werevisible. The pattern of callose deposition was assessed inwild-type and tramgap mutant ovules to determinewhether two or more enlarged cells in tramgap ovulesshow MMC-specific callose deposition. Ovules of thewild type showed uniform callose deposition in theMMC before the initiation of meiosis (Fig. 8, C andD); after meiosis, callose deposition was observed in
Table IV. Frequency of FGs at different stages of development at anthesis in wild-type, tramgap1-1, andtramgap1-2 mutant lines
Values shown are percentages. DE, Degenerating; N, total number of ovules examined.
Figure 5. Comparative analysis of anther devel-opment in the wild type and homozygous tramgapmutants. Anther development is shown in the wildtype (top row), in homozygous tramgap1-1 (mid-dle row), and in homozygous tramgap1-2 (bottomrow). A, Cross section of wild-type anther at stage9 showing proper development of microsporesand tapetum. B, Cross section of homozygoustramgap1-1 anther at stage 9 showing defectivemicrospores and tapetum. C, Cross section of ho-mozygous tramgap1-2 anther at stage 9 showingdefective microspores and tapetum. D, Wild typeanther at stage 11 showing mature pollen anddegenerated tapetum. E, Sterile and fertile pollenand partly consumed tapetum in homozygoustramgap1-1. F, Sterile and fertile pollen and partlyconsumed tapetum in homozygous tramgap1-2.G, Wild-type anther at stage 13 showing dehis-cence and release of fertile pollen. H, Fertile andsterile pollen in homozygous tramgap1-1 at stage13. I, Fertile and sterile pollen in homozygoustramgap1-1 at stage 13. DP, Defective pollen; MP,mature pollen; T, tapetum.
transverse walls between the functional megaspore andits degenerated sister cells (Fig. 8, E and F). In tramgap1-2ovules, faint patches of callose deposition were seen inenlarged abnormal gametic cells (Fig. 8, G and H).However, in tramgap mutant ovules at postmeioticstages, callose deposition was observed in MMC as wellas in the nucellar region (Fig. 8, I and J). Ovules withseveral enlarged cells in the tramgap mutant showedcallose deposition in multiple cells (Fig. 8, K and L).These results indicate that all four meiotic products hadsurvived in some of the ovules and that TRAMGaP isessential for restricting the identity and differentiation ofMMC to a single subepidermal cell in premeiotic ovules,which may be achieved in combination with AGO-9.
AGOs and katanin-like proteins participate in thetranslational repression of genes guided by siRNA/microRNA (miRNA; Brodersen et al., 2008). An in silicosearch at AthaMap (www.athamap.de/search.php?restriction=0&chromosome=5&pos=9226100) revealedthat TRAMGaP is a small RNA-regulated gene with onemiRNA (MIR777) targeting the 59 untranslated regionand 30 siRNAs matching the TRAMGaP gene. A ma-jority of these siRNAs had overlapping sequences thatcorresponded to the fourth intron of TRAMGaP(Supplemental Fig. S12). Furthermore, two-thirds ofthese siRNAs were associated with AGO4. Thus,TRAMGaP mutation is expected to affect siRNA andtargetmRNAstoichiometry, leading to pleiotropic effects.
DISCUSSION
Intercellular communications between sporophyticand gametophytic cells are critical for cell specificationand proper development during the alternation ofgenerations (Chevalier et al., 2011). Although a numberof genes involved in embryo sac development havebeen identified in Arabidopsis, the signaling pathwaysand gene interactions are far from clear. In animals,TRAF proteins serve as adaptors in signal transductioninvolving membrane-bound TNF-R. Very few of thelarge number of TRAF-like genes that have been an-notated in Arabidopsis have been investigated so far.Considering that TRAF-like genes in Arabidopsis oc-cur in a cluster and share a high degree of homology(Cosson et al., 2010), functional redundancy mightprevent the ready manifestation of mutant phenotypes.To our knowledge, this is the first report to demonstratethat TRAF-like genes are critical to reproductive de-velopment in plants.
We studied two T-DNA insertion mutants each dis-rupting different MATH domains of the TRAMGaP gene.Both showed comparable altered phenotypes in hetero-zygous plants, probably owing to haploinsufficiency. Theexpression pattern of TRAMGaP assessed by reportergene assay showed GUS expression in the developinggametophytes and in the surrounding sporophytic tissue.Furthermore, crossing experiments and pollen viability
Figure 6. Analysis of pollen viability, integrity, and structure in the wild type and homozygous tramgap1-1 and tramgap1-2mutants. A, DIC image of anther from the wild type showing normal pollen. B, DIC image of anther from a homozygous tramgap1mutant showing defective pollen. C,Wild type anther stainedwith Alexander’s stain showing fully viable pollen. D, Homozygoustramgap1-1 anther stained with Alexander’s stain showing empty and nonviable pollen (arrows) along with viable pollen. E,Scanning electron micrograph showing normal, elliptical wild-type pollen with proper reticulate exine. F, Scanning electronmicrograph showing abnormal, shriveled, irregularly shaped tramgap1 pollen with smooth exine. G, DAPI-stained wild-typepollen showing trinucleate pollen. H, Pollen from a homozygous tramgap1-1mutant with zero, one, two, or three nuclei. Insets inE and F are closeup views of a single pollen grain showing defective pollen wall. Bars = 20 mm.
studies clearly indicated that the development of wild-type gametes also was affected adversely in heterozy-gous mutants. Thus, the tramgap mutation was found tohave both sporophytic and gametophytic effects. Never-theless, the tramgap mutation was not completely lethaland some homozygous plants were obtained, probablybecause the overexpression of some closely relatedparalogs compensated to some extent for the loss oftramgap expression.TRAFs regulate several functions of the TNF-R su-
perfamily, apparently by linking the cytosolic domainof the receptors to downstream protein kinases orubiquitin ligases, and the trimerization of the MATHdomain of TRAFs is essential for the binding of otherdownstream signaling proteins (Arch et al., 1998;Wallach et al., 1999; Deng et al., 2000). In animals, theTNF-R superfamily is involved in a wide range of bio-logical functions, such as adaptive and innate immu-nity, embryonic development, stress response, andbone metabolism, through the induction of cell activa-tion, cell survival, and antiapoptotic functions mostlymediated by the family of TRAFs (Park et al., 2000;Chung et al., 2002). Therefore, disruption of this do-main is expected to abolish gene function. This expec-tation was strengthened by the observation that thetramgap1-2 mutation that led to the loss of both MATHdomains produced a more pronounced phenotype. TheMATH domain of MEL-26 (a substrate adaptor) inter-acts with specific substrates such as MEI-1 or katanin toassociate with the CUL-3 complex (Srayko et al., 2000;Kurz et al., 2002; Pintard et al., 2003; Bowerman andKurz, 2006). These findings indicate that the MATHdomain interacts directlywith the KATANINprotein inthe CRL complex. Burk et al. (2001) reported that a
mutation in AtKTN1, the Arabidopsis ortholog ofkatanin, results in siliques that are 30% to 80% shorterthan normal siliques, a feature that was also observed inthe tramgap mutants. These results strongly implicateMATH as an interacting partner for the katanin-likeprotein. The BTB/SPOP adaptor proteins of the CUL-3 complex have been shown to bind to the substrate atone end through the MATH domain and to the CRLcomplex at the other end through the BTB domain(Pintard et al., 2003; Hua and Vierstra, 2011; Zhanget al., 2014). The two MATH domains in At5g26290might behave as the BTB/SPOP-like protein of the CRLcomplex. Quaternary structure analysis of TRAMGaPalso showed that one of the MATH domains was aTRAF-like protein whereas the other was similar to theSPOP. Therefore, we speculate that TRAMGaP worksas a substrate adaptor protein of the CRL complex.
In C. elegans, KATANIN localizes to the spindle andchromosomes during meiosis, and ubiquitin-ligase ac-tivity of the CUL-3 complex is required to degradeKATANIN after meiosis, which is essential for assem-bling a functional mitotic spindle leading to the meiosis-to-mitosis transition (Pintard et al., 2003). The persistenceof meiotic products in ovules at anthesis clearly indi-cates that meiosis-to-mitosis transition signaling isdisrupted in tramgap mutants. Moreover, katanin dis-ruption has been shown to increase the polypeptidelevels of several small RNA-regulated genes, includingargonautes, without a corresponding change in theabundance of their mRNAs (Ma et al., 2013). A recentstudy by Gao et al. (2014) showed that miRNAs play animportant role in regulating the TRAF-mediated NF-kBsignaling pathway. Classification of At5g26290 as asiRNA target gene (AthaMap), the association of siRNAs
Table V. DAPI analysis of pollen of tramgap mutants
Genotype No. of Pollen Grains ExaminedPercentage of Pollen Showing
aMeans of samples differ from the wild type at P , 0.01.
Table VI. qRT-PCR analysis of TRAMGaP (At5g26290) and its close paralogs at various stages of flowerdevelopment in the wild type and the SALK mutant line Numbers indicate log fold differences.
GeneWild Type SALK
5–9a 10–12 13–14 15–18 5–9 10–12 13–14 15–18
At5g26260 24.24 24.74 25.87 28.54 24.46 24.84 26.45 27.08At5g26280 26.08 24.02 24.26 29.00 24.61 22.11 22.35 26.97At5g26290b 0.00 27.05 24.19 No CT No CT No CT No CT No CTAt5g26300 28.42 23.05 22.54 24.17 No CT 23.64 No CT 23.24At5g26320 27.14 22.63 20.23 22.14 24.17 23.65 23.70 22.69
aFlower stages 5–9, 10–12, 13–14, and 15–18 correspond to microsporogenesis, megasporogenesis,anthesis, and postfertilization stages, respectively. bAt5g26290 (i.e. TRAMGaP) is taken as the cali-brator. No CT means RT-PCR amplification did not reach the threshold level
corresponding to the TRAMGaP gene with AGO4, andthe down-regulation of different argonaute members intramgap mutants suggest that disturbance of one genewould affect the other. Olmedo-Monfil et al. (2010) andTucker et al. (2012) showed that argonaute proteins arecritical to signaling during the development of gameto-genic progenitor cells (mitotic-to-meiotic transition) andduring gametogenesis,whereinAGO9 is confinedmainlyto the L1 layer of the developing ovule but passes on itssignal to gametogenic progenitor cells to form functionalmegaspores; however, an AGO gene from rice (MEL1)was reported to express itself in MMC (Nonomura et al.,2007). Both AGO5 and AGO104were expressed in the L1layer and in the nucellus, suggesting signaling from thesporophytic tissue of the nucellus to the developinggermline (Singh et al., 2011; Tucker et al., 2012). TRAMGaPshares the expression pattern of the above-mentionedargonautes in that it is expressed in the L1 layer, in thenucellus, and in MMC (Figs. 3 and 8). As an adaptormolecule, TRAMGaP probably acts as a linker or as acarrier to transmit signals from sporophytic tissues togametophytic tissues, thereby affecting gametophytedevelopment. The occurrence of AGO9-like and relatedphenotypes (Figs. 4 and 8) at the stage of gametogenicprogenitor cell differentiation points to TRAMGaP asan important member of this signaling pathway. This
assumption is further supported by the altered ex-pression of genes involved in siRNA biogenesis inconjunction with argonautes (Supplemental Table S3).
The overrepresentation of hormone-related genesin microarray (Supplemental Table S2), their down-regulation at the transcription level (Fig. 7; SupplementalTable S3), and the report that At5g26290 (TRAMGaP) is acytokinin-responsive gene (Bhargava et al., 2013) lendfurther support to the assumption that TRAMGaP acts bydirect or indirect bindingof katanin-like proteins to controlhomeostasis in hormonal signals. GA signaling is routedthrough the CRL complex, leading to the ubiquitination ofDELLA proteins (Plackett et al., 2011). These proteins arenegatively regulated by MYB-like genes, which, in turn,enlistAGAMOUS-like genes to control downstreamgenesinvolved in male gametogenesis (Tang et al., 2010). Cyto-kinins are crucial to female gametogenesis and induceArabidopsis Response Regulators (Cheng et al., 2013),which are involved in programmed cell death and thespecification of functional megaspores in the FG in Ara-bidopsis (Vescovi et al., 2012; Cheng et al., 2013). Similarly,auxins are critical to the sporophytic-to-gametophytic celltransition as well as at later stages of ovule development,namely cellularization and speciation of the seven-celledembryo sac (Schmidt et al., 2015). Furthermore, F-boxproteins act through the CRL complex and are known to
Figure 7. qRT-PCR analysis of selected reproductive pathway genes in the wild type (WT) and the tramgap1-2mutant. Mutationin TRAMGaP leads to down-regulation of many genes involved in different development-related pathways. qRT-PCR analysis forkey genes involved in reproductive development, including pathway genes related to RNA-mediated development, CRL com-plex, ovule and pollen development, and lipid and hormones, showed differential expression in the homozygous tramgap1-2mutant. Most of the genes were down-regulated in the mutant.
regulate the cell cycle machinery to promote male germcell division in Arabidopsis (Borg et al., 2009). The pro-gression to reproductive phase, acquisition, and the re-striction of reproductive cell fate during the somatic-to-gametic transition involve many different pathwayssuch as hormonal pathways, siRNA-mediated regulation,posttranscriptional controlmechanisms, and regulation bytranscription factors (Schmidt et al., 2015). The overrep-resentation of genes related to different hormones, kinases,and components of the CRL complex in microarray anal-ysis (Supplemental Table S2), the altered expression ofrepresentative key genes recorded in qRT-PCR (Fig. 7;Supplemental Table S3), and the phenotypes observed atdifferent stages of reproductive development indicateTRAMGaP to be an important molecular link that coor-dinates the development of male and female gametes inArabidopsis.In animals, TRAF proteins mediate apoptosis and cell
division (Arch et al., 1998), and the observed pheno-types during the development of the ovule and pollenin Arabidopsis suggest conservation of this function inplants as well. Programmed cell death of the tapetumand the formation of exine are closely interlinked interms of the production and accumulation of materialsrequired for the formation of sporopollenin and itscorrect deposition on the pollen surface (Vizcay-Barrena and Wilson, 2006; Zhou et al., 2012). Defectsin exine formation and the delayed programmed celldeath of tapetal cells in tramgapmutants (Figs. 3, 5, and6) and TRAMGaP gene expression in tapetum cells ofthe wild type (Fig. 3) clearly link programmed cell
death to TRAMGaP. Moreover, according to eFPbrowser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi), most of the phenylpropanoid and lipidsynthesis genes are expressed during flower develop-mental stages 10 and 11 (prefertilization stages). Theresults of microarray analysis and qRT-PCR in theseexperiments showed that a number of genes related tothe above pathways were down-regulated in the tram-gap mutant and may account for the defects in pro-grammed cell death of the tapetum.
Furthermore, we also observed several defects inmegaspore and microspore mitoses. Among the genesinvolved in the development of the gametophyte or inthe early stages of seed development, the expression ofSWA1 and PHE1 was down-regulated in tramgap mu-tants as compared with that in the wild type. AlthoughSWA1 is expressed in several types of tissue, it isstrongly expressed in anthers, in functional mega-spores, and in the embryo sac (Shi et al., 2005). Fur-thermore, SWA1 mutation leads to the development ofthe embryo sac being arrested at the two-, four-, oreight-cell stage. The differential expression of key genesregulating cell division during the male and femalemitotic phase of growth, as shown by the microarrayand qRT-PCR analyses, further suggests the orchestra-tion of key genes controlling male and female gameto-genesis by TRAMGaP through a complex network ofinteracting partners. Such key genes include ACT8,PROLIFERA, AN3, ARGONAUTES, FIE, PHE1, LIS,RBR1, and MADS-BOX (Holding and Springer, 2002;Anastasiou et al., 2007; Kapoor et al., 2008; Drews and
Figure 8. Callose deposition in developing ovules of the wild type and homozygous tramgap mutants of Arabidopsis. A, Post-meiotic tramgap1-1 ovule showing abnormal gametic cells (AGC) adjacent to a degeneratedmegaspore (arrows) and a functionalmegaspore (FM). B, Postmeiotic tramgap1-2 ovule showing abnormal gametic cells adjacent to a degenerated megaspore (as-terisk) and a functionalmegaspore. C, DIC image of a premeiotic wild-type ovule showing a singleMMC.D, Fluorescent image ofthe ovule in C showing uniform callose deposition on the MMC. Callose is seen on transverse walls between the functionalmegaspore and degenerated sister cells. E, DIC image of a premeiotic tramgap1-2 ovule showing twoMMCs. F, Fluorescent imageof the ovule in E showing faint callose patches in both MMCs. G, DIC image of a postmeiotic tramgap1-2 ovule showing MMCand surrounding tissue. H, Fluorescent image of the ovule in G showing callose patches in MMC and also in surrounding cells. I,DIC image of a postmeiotic tramgap1-2 ovule showing multiple megaspores. J, Fluorescent image of the ovule in I showingpatches of callose in a number of cells. K, DIC image of a postmeiotic tramgap1-2 ovule showing multiple megaspores. L,Fluorescent image of the ovule in K showing callose patches in a number of cells. Percentages are the frequencies that occur in therespective phenotypes of the mutant.
Koltunow, 2011; Tucker et al., 2012). The protein-proteininteraction network of differentially expressed genes inmicroarray and their gene ontology (Supplemental Fig.S11) also suggest a similar role for TRAMGaP.
Based on the phenotypes, microarray, and qRT-PCRanalysis, we propose a TRAF-mediated signal trans-duction pathway model (Supplemental Fig. S13) to ex-plain the probable role of TRAMGaP in reproductivedevelopment. Ubiquitination coupled with kinase-mediated signaling is a prerequisite to activation of theubiquitin-proteasome system for hormone-mediatedplant growth and development (Sadanandom et al.,2012). The interaction of kataninwith theMATHdomainof the CRL complex signals the regulation of differenttranscription factors mediated through hormonal ho-meostasis, which, in turn, involves precisely controlledorchestration of the differential expression of genes. Chenet al. (2013) showed that TRAFs are negatively regulatedby E3 ligase components involving the F-box protein. Thehormonal signals were subjected to controlled synthesisand degradation through SCF-like F-box proteins, which,in turn, are regulated by DELLA proteins. Our resultssupport the involvement of TRAMGaP in gametogenesis-related gene expression and regulation through ubiquitin-mediated signal transduction. The SCF complexes areinvolved in many cellular and developmental processesin plants (Choi et al., 2014). A complete loss of theirfunction results in an embryo-lethal phenotype, whereasthe hypomorphic mutants show sterility and pleiotropicdefects affecting flower development; auxin, cytokinin,GA3, and jasmonate signaling; progression of meiosis inmale gametes; and embryogenesis (Choi et al., 2014).Furthermore, the defects in the signaling complex changethe expression of key genes that regulate different devel-opmental stages in male and female gametogenesis.
Thus, we conclude that the TRAMGaP protein playsan important role in reproductive development byacting as an adaptor molecule of a complex signalinghub that integrates various transcription factors, hor-monal signals, and some key regulatory genes of maleand female gametogenesis. Our findings open the wayto test various possible interactions between TRAMGaPand other proteins to establish a clear molecular path-way of the complex process of gametogenesis in plants.
MATERIALS AND METHODS
Plant Growth and Hybridization
While screening a population of Arabidopsis (Arabidopsis thaliana) T-DNApromoter trap (Columbia-0) developed in house, we noticed a line showingonly partial seed set. Surface-sterilized seeds of that line and of the SALK line(SALK_0146328) were sown on Murashige and Skoog (1962) medium con-taining kanamycin (50 mg L21), and 16-d-old seedlings were transferred to potsfilled with a mixture of vermiculite, peat moss, and perlite in equal proportionsby volume. The plants were raised in a growth chamber (Conviron; modelAdaptis A-1000) with 16 h of light alternatingwith 8 h of darkness at 21°C6 1°Cand 70% relative humidity. For transmission studies of the mutant alleles,flower buds were emasculated manually 1 d before anthesis and covered withbags made of waxed paper. The emasculated buds were pollinated the next daywith fresh pollen from selected plants and covered again. Siliques were har-vested at maturity, hybrid seeds were collected, and the progeny were raised as
above. Themarker line ET884was a gift fromUeli Grossniklaus, andDD65:GFPwas obtained from G.C. Pagnussat’s laboratory and analyzed as described byPagnussat et al. (2007).
Phylogenetic Analysis
The third largest cluster of TRAF-like genes ofArabidopsiswas alignedusingClustalW (MEGA 6.0; Tamura et al., 2013; http://www.ebi.ac.uk/Tools/msa/clustalw2/). The maximum parsimony programwas used for tree constructionwith the 1,000 bootstrap test to judge the robustness of branches being clus-tered. The protein AML2 (At2g42890) was used as an outgroup because it isparalogous to TRAMGaP, a meiotic gene involved in a similar function, andshowed a minimal level of homology (28%) throughout the protein sequence ofTRAMGaP (Kaur et al., 2006).
DNA/RNA Isolation and Identification of the T-DNAInsertion Site
DNAandRNAwere isolated from leaf or inflorescence samples as describedearlier (Pratibha et al., 2013, 2017). The T-DNA insertion site in the mutant linewas identified using genome walking (Pratibha et al., 2013). To identify thehomozygous mutant lines, primers were designed from the T-DNA region andthe flanking Arabidopsis chromosomal region. Homozygous and heterozygousmutant plants were separated based on the presence or absence of specific PCRfragments (Supplemental Fig. S1). Nucleotide sequences of the primers used inthis study are given in Supplemental Table S4.
Mutant Complementation
The coding sequence of the At5g26290 gene was amplified with specificprimers (Supplemental Table S4) havingNcoI and BglII restriction sites at the 59ends of the forward and reverse primers, respectively, and cloned into apCAMBIA1302 vector downstream of the cauliflower mosaic virus 35S pro-moter. Homozygous mutant plants carrying the T-DNA insertion in theAt5g26290 gene were transformed with Agrobacterium tumefaciens containingthe above gene cassette, and T1 plants were selected on Murashige and Skoogagar plates supplemented with hygromycin (20 mg mL21). Hygromycin-resistant seedlings were transferred to pots and raised to maturity.
Promoter Characterization
A 1.5-kb fragment upstream of the At5g26290 coding sequence was ampli-fied by PCR using specific primers (Supplemental Table S4) and cloned into thepORE plant binary vector (Coutu et al., 2007) between HindIII and SacI re-striction sites to drive the uidA reporter gene. Wild-type Arabidopsis plantswere transformed with A. tumefaciens containing the above promoter-reporterconstruct, and T1 plants were analyzed for GUS expression using a histo-chemical assay (Jefferson et al., 1987). Tissues cleared with 70% (v/v) ethanolwere observed directly with a microscope (Axio Imager 1000; Carl Zeiss).
Alexander’s Staining
Flower buds were incubated overnight in ethanol at 4°C; the anthers weredissected on a slide and incubated in Alexander’s stain (Alexander, 1969),mounted with a coverslip, kept for 30 min at room temperature, and observedwith the microscope with DIC optics through a 403 objective.
Whole-Mount Preparation of Ovules and Anthers
Thedevelopment of the ovulewas studiedusingwholemounts after clearingthe inflorescence inmethyl benzoate as described by Siddiqi et al. (2000). Briefly,flowers were fixed in 3.7% (v/v) formalin, 5% glacial acetic acid, and 50% (v/v)ethanol (v/v) overnight at 4°C and dehydrated by passing through a series ofacetone solutions. The dehydrated tissues were cleared for 2 h in methyl benzoate.Ovules were dissected on a slide using a stereo dissecting microscope, mountedwith a coverslip, and observed with the microscope as described above.
The development of pollen was studied by fixing the inflorescence in for-malin, glacial acetic acid, and 50% (v/v) ethanol (1:1:18) for 2 d at room tem-perature. The samples were dehydrated subsequently in a tertiary butyl alcohol
series (Jensen, 1962), embedded in paraffin (melting point, 58°C–60°C), and 8- to10-mm-thick sections were cut using a Finesee microtome. The sections weredewaxed and stained with 0.1% (w/v) Toluidine Blue in water followed byrinsing in clove oil to remove haziness, mounted in Canada balsam, and ex-amined using conventional bright-field microscopy.
For DAPI staining, samples were stained with 0.1% (w/v) DAPI in 0.1 M
phosphate buffer, pH 7, and photomicrographs were taken under UV illumi-nation. To examine the architecture of the pollen wall and the patterns on theexine, pollen grains were sputtered on a double-sided rubber tape, coated withcarbon using a sputter coater (E1010; Hitachi), and viewed with a HitachiS-3400N field emission scanning electron microscope using an acceleratingvoltage of 30 kV.
Total RNA Isolation
Total RNA (50–100 mg) was isolated from the wild type and from the ho-mozygous SALK mutant line using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and subjected to on-column DNase (Sigma-Aldrich) treatment toremove any contaminating DNA. The quality of RNA samples was analyzed ona 1.2% (w/v) denaturing agarose gel and quantified using NanoDrop2000 (Thermo Scientific), and the samples were stored at280°C until required.
Quantitative Real-Time PCR Analysis
Total RNA (1 mg) was converted to single-stranded cDNA using InvitrogenSuperScript III Reverse Transcriptase (Thermo Fisher) by following the man-ufacturer’s protocol, and qRT-PCR analysis was performed in an Mx3005Psystem (Stratagene and Agilent Technologies) using the KAPA SYBR FASTqPCR Kit (KAPA Biosystems). Total cDNA was diluted to ;25 ng mL21, and atotal of 100 ng was used in a 10-mL reaction mixture. For each reaction, threetechnical replicates were used along with a no-template control to check forcontaminants. The following thermal cycling program was used for all qRT-PCRs: 3 min at 95°C (enzyme activation), 3 s at 95°C (denaturation), and 30 s at60°C (annealing/extension) for 40 cycles, which includes data acquisition. Fi-nally, a dissociation curve analysis was performed from 65°C to 95°C in in-crements of 0.5°C, each lasting for 5 s, to confirm the presence of a specificproduct. qRT-PCR primers were designed using standard parameters availableat http://eu.idtdna.com/scitools/Applications/RealTimePCR (SupplementalTable S4). The concentration of ACT2 (At3g18780) was used to normalize geneexpression in different samples, and the expression of TRAMGaP (At5g26290)in wild-type flower buds (stages 5–9) was used to calibrate the data in othersamples. Log fold changes in expression values were calculated using the 22DDCT
method (Livak and Schmittgen, 2001).
Microarray Analysis
Global gene expression analysis and identification of the key genes show-ing differential regulation in the tramgap1-2 mutant were carried out usingGeneChip Arabidopsis ATH1 Genome Array (Affymetrix) representing ;24,000genes. The homozygous tramgap1-2mutant line was used for comparison withthe wild type. A total of three technical replicates were used for each sampleanalyzed. From each wild-type and mutant plant, samples were collected fromwhole inflorescence, which included floral buds at stages 5 to 18. Total RNAwas isolated as described above. For labeling, total RNA (100–250 ng) wasamplified and labeled in three independent reactions using the 39 IVT expresslabeling kit (Affymetrix). The biotinylated RNA samples were fragmented andhybridized to the GeneChip Arabidopsis Genome Array. The arrays werewashed using an Affymetrix GeneChip fluidic station 400 and scanned using aHewlett-Packard Gene Array Scanner G2500A. CEL files were generated usingthe combined console software (Affymetrix). Data from the chips showing 0.9or greater Pearson correlation coefficient between replicates were included inthe analysis.
TheCELfileswere loaded intoGeneSpringGX11 (AgilentTechnologies), andthe robust multi-array average (RMA) algorithm was applied to normalize thedata. Quality control (principal component and correlation analyses) was per-formed among the replicates to detect batch effects or other random effects. Toidentify the differentially expressed genes, a two-way ANOVA was used, andthe computed P values were corrected by the Benjamini-Hochberg (false dis-covery rate) multiple testing correction method. Furthermore, probe identifierssatisfying two conditions, namely P# 0.05 and a 2-log fold or greater change inexpression level, were shortlisted for detailed analysis.
The differentially expressed genes were used to prepare the protein-proteininteraction network of Arabidopsis by using Cytoscape, and putative functionsof the clustered genes were investigated using ClueGO and REVIGO (Supeket al., 2011).
Accession Number
The Arabidopsis Information Resource accession number for TRAMGaPgene is At5g26290.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Identification of the T-DNA insertion in the tram-gap1-1 mutant by genome walking.
Supplemental Figure S2. Nucleotide and amino acid sequences ofTRAMGaP.
Supplemental Figure S3. Phylogenetic tree generated with known TRAFgenes from plants and animals.
Supplemental Figure S4. Phylogenetic analysis of TRAF genes of Arabi-dopsis.
Supplemental Figure S5. Cluster alignment of TRAF-like genes of Arabi-dopsis showing conservation of the At5g26290 protein with its neigh-boring genes of the family.
Supplemental Figure S6. Prediction of the secondary structure of theAt5g26290 protein.
Supplemental Figure S7. Prediction of the quaternary structure of theAt5g26290 protein.
Supplemental Figure S9. Expression profile of the TRAMGaP gene.
Supplemental Figure S10. Abnormal ovule phenotypes in tramgap mu-tants.
Supplemental Figure S11. REVIGO of differentially expressed genes inmicroarray analysis of tramgap1-2 during flower development corre-sponding to stages 5 to 9.
Supplemental Figure S12. Base pairing between siRNAs or miRNA se-quences with the At5g26290 gene.
Supplemental Figure S13. Possible TRAMGaP-mediated signal transduc-tion during male gametophyte and FG development in Arabidopsis.
Supplemental Table S1. qRT-PCR of TRAMGaP in various tissues.
Supplemental Table S2. Microarray analysis of the tramgap1-2/tramgap1-2mutant.
Supplemental Table S3. qRT-PCR of key genes with altered expression intramgap1-2/tramgap1-2 at preanthesis and postanthesis.
Supplemental Table S4. Details of primers used in this study.
ACKNOWLEDGMENTS
The help extended by Dr. Avnesh Kumari in scanning electron microscopyanalysis is gratefully acknowledged.
Received February 23, 2017; accepted September 17, 2017; published September22, 2017.
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