Interactions between tassel seed genes and other sex determining genes in maize

The tassel seed mutations of maize cause sex reversal of the florets of the tassel, such that the normally staminate florets develop pistils. Although these mutations have been recognized for many years, little is known about how they act. We have tested the hypothesis that the tassel seed genes interact directly with each other and with other genes controlling sex determination in a single genetic pathway by the construction and analysis of double mutants. On the basis of the phenotypes of the double mutants, the tassel seed mutations were placed into two groups: ts1, ts2, Ts5 and ts4, Ts6. Both groups of tassel seed mutations were additive with the masculinizing mutation dwarf, indicating independent modes of action. Interactions of tassel seed mutations with silkless varied, allowing the ordering of the action of the various tassel seed mutations relative to silkless. Both groups of tassel seed mutations were epistatic with regard to sex expression to mutations that alter both architecture of the plant and distribution of male and female florets, Teopod 1, terminal ear, and teosinte branched. Thus, there are at least two separate genetic pathways that control the sex of florets in maize tassels. In addition, analysis of double mutants revealec that all tassel seed genes tested play a role in the regulation of flower morphogenesis as well as pistil suppression. © 1994 Wiley-Liss, Inc.     

Joint‐linkage mapping and GWAS reveal extensive genetic loci that regulate male inflorescence size in maize

Both insufficient and excessive male inflorescence size leads to a reduction in maize yield. Knowledge of the genetic architecture of male inflorescence is essential to achieve the optimum inflorescence size for maize breeding. In this study, we used approximately eight thousand inbreds, including both linkage populations and association populations, to dissect the genetic architecture of male inflorescence. The linkage populations include 25 families developed in the U.S. and 11 families developed in China. Each family contains approximately 200 recombinant inbred lines (RILs). The association populations include approximately 1000 diverse lines from the U.S. and China. All inbreds were genotyped by either sequencing or microarray. Inflorescence size was measured as the tassel primary branch number (TBN) and tassel length (TL). A total of 125 quantitative trait loci (QTLs) were identified (63 for TBN, 62 for TL) through linkage analyses. In addition, 965 quantitative trait nucleotides (QTNs) were identified through genomewide study (GWAS) at a bootstrap posterior probability (BPP) above a 5% threshold. These QTLs/QTNs include 24 known genes that were cloned using mutants, for example Ramosa3 (ra3), Thick tassel dwarf1 (td1), tasselseed2 (ts2), liguleless2 (lg2), ramosa1 (ra1), barren stalk1 (ba1), branch silkless1 (bd1) and tasselseed6 (ts6). The newly identified genes encode a zinc transporter (e.g. GRMZM5G838098 and GRMZM2G047762), the adapt in terminal region protein (e.g. GRMZM5G885628), O‐methyl‐transferase (e.g. GRMZM2G147491), helix‐loop‐helix (HLH) DNA‐binding proteins (e.g. GRMZM2G414252 and GRMZM2G042895) and an SBP‐box protein (e.g. GRMZM2G058588). These results provide extensive genetic information to dissect the genetic architecture of inflorescence size for the improvement of maize yield.     

Experimental Analysis of Tassel Development in the Maize Mutant Tassel Seed 6

The maize (Zea mays L.) mutation Tassel seed 6 (Ts6) disrupts both sex determination in the tassel and the pattern of branching in inflorescences. This results in the formation of supernumerary florets in tassels and ears and in the development of pistils in tassel florets where they are normally aborted. A developmental analysis indicated that extra florets in Ts6 inflorescences are most likely the result of delayed determinacy in spikelet meristems, which then initiate additional floret meristems rather than initiating floral organs as in wild type. I have used culturing experiments to assay whether delayed determinacy of Ts6 mutant tassels is reflected in an altered timing of specific determination events. Length of the tassel was used as a developmental marker. These experiments showed that although Ts6 tassels elongate much more slowly than wild type, both mutant and wild-type tassels gained the ability to form flowers with organs of normal morphology in culture at the same time. In situ hybridization patterns of expression of the maize gene Kn, which is normally expressed in shoot meristems and not in determinate lateral organs, confirmed that additional meristems, rather than lateral organs, are initiated by spikelet meristems in Ts6 tassels.     

DEVELOPMENT OF TASSEL SEED 2 INFLORESCENCES IN MAIZE

The normal pattern of maize floral development of staminate florets on the terminal inflorescence (tassel) and pistillate florets on the lateral inflorescences (ears) is disrupted by the recessive mutation tassel seed 2. Tassel seed 2 mutant plants develop pistillate florets instead of staminate florets in the tassel. In addition, the ears of tassel seed 2 plants display irregular rowing of kernels due to the development of the normally suppressed lower floret of each spikelet. The morphology of tassel and ear florets of the recessive maize mutant tassel seed 2 has been compared to those of wild-type maize through development. We have identified the earliest stages at which morphological signs of sex differentiation are evident. We find that sex determination occurs during the same stage on tassel and ear development. Early postsex determination morphology of florets in wild-type ears and in tassel seed 2 tassels and ears is identical.     

Class II tassel seed mutations provide evidence for multiple types of inflorescence meristems in maize (Poaceae)

The tassel seed mutations ts4 and Ts6 of maize cause irregular branching in its inflorescences, tassels, and ears, in addition to feminization of the tassel due to the failure to abort pistils. A comparison of the development of mutant and wild-type tassels and ears using scanning electron microscopy reveals that at least four reproductive meristem types can be identified in maize: the inflorescence meristem, the spikelet pair meristem, the spikelet meristem, and the floret meristem. ts4 and Ts6 mutations affect the fate of specific reproductive meristems in both tassels and ears. ts4 mutants fail to form spikelet meristems from spikelet pair meristems. Ts6 mutants are delayed in the conversion of certain spikelet meristems into floret meristems. Once floret meristems are established in both of these mutants, they form florets that appear normal but fail to undergo pistil abortion in the tassel. The abnormal branching associated with each mutant is suppressed at the base of ears, permitting the formation of normal, fertile spikelets. The classification of the different types of reproductive meristems will be useful in interpretation of gene expression patterns in maize. It also provides a framework for understanding meristem functions that can be varied to diversify inflorescence architectures in the Gramineae.     

Use of homeotic mutation tasselseed2 for investigation of the action of maize meiotic genes during micro-and megasporogenesis

The manifestation of the ms43 maize meiotic mutation in the megasporogenesis of ts2 ms43 double mutants has been studied. Combined genetical and cytological analysis of the progeny of diheterozygote selfing showed that the ms43 mei gene was not microsporogenesis-specific. The manifestation of ms43 in megasporogenesis of the double mutants proved to be affected by the ts2 mutation. It prevented formation of the phenotype characteristic of ms43 and distorted early developmental stages of the entire ovule. It is the first study of megasporogenesis in tasselseed2 transformed tassels. Cytological data on the afd1 mutation in single and double mutants are presented. Possible mechanisms of the interaction between the ms43 and ts2 mutations are discussed.     

The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine‐mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu‐induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one‐carbon (C1) metabolism to polyglutamylate substrates of folate‐dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS‐deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild‐type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.     

Use of homeotic mutation tasselseed2 for investigation of the action of maize meiotic genes during micro- and megasporogenesis

The manifestation of the ms43 maize meiotic mutation in the megasporogenesis of ts2 ms43 double mutants has been studied. Combined genetical and cytological analysis of the progeny of diheterozygote selfing showed that the ms43 mei gene was not microsporogenesis-specific. The manifestation of ms43 in megasporogenesis of the double mutants proved to be affected by the ts2 mutation. It prevented formation of the phenotype characteristic of ms43 and distorted early developmental stages of the entire ovule. It is the first study of megasporogenesis in tasselseed2 mutant tassels. Cytological data on the afd1 mutation in single and double mutants are presented. Possible mechanisms of the interaction between the ms43 and ts2 mutations are discussed.     

Regulation of sex determination in maize

Maize develops separate male and female flowers in different locations on a single plant. Male flowers develop at the tip of the shoot in the tassel, and female flowers develop on the ears, which terminate short branches. The development of male flowers in tassels and female flowers in ears is the result of selective abortion of pistils or stamens, respectively, in developing florets. Genetic analysis has shown that stamen abortion and pistil abortion are under the control of two different genetic pathways. Local levels of the plant hormone gibberellic acid determine whether or not stamens are suppressed. Pistil abortion is under the regulation of the tassel seed genes, one of which has been shown to encode a short-chain alcohol dehydrogenase. The tassel seed genes play a role in regulating the fate of inflorescence meristems as well as pistil primordium fate.     

玉米性别决定的激素调控

玉米(Zea mays)属典型的雌雄异花植物, 单性花的形成经历了复杂的性别决定过程。通过雄穗小花和雌穗下位花的雌蕊原基以及雌穗小花雄蕊原基的选择性败育(或退化), 玉米最终形成正常的雌雄同株单性花。相关突变体的研究揭示, 玉米性别决定涉及选择性细胞死亡、细胞保护及信号转导等复杂的过程。其中, 植物激素信号的调控在玉米性别决定过程中处于核心地位。最近的研究表明, 赤霉素、细胞分裂素和茉莉酸类物质参与调控玉米性别决定过程。该文结合最新研究成果, 综述了植物激素在玉米性别决定中的作用及其调控途径, 同时提出了研究中存在的问题, 并对该领域未来的研究方向进行了展望。     

两个相关基因表达量和SNP与玉米雄穗大小相关

玉米雄穗通常较发达,散粉量大于授粉需要,过量消耗能量会影响光合产物向果穗的分配,过于发达的雄穗还会影响群体透光性、降低光合效率。生产实践和育种研究证明,由于雄穗大小与玉米籽粒产量负相关,因此成为品种选育的间接选择指标。该研究根据前人的报道,从11个雄穗大小不同的玉米自交系中扩增角蛋白相关蛋白基因KAP5-4和受体样蛋白激酶基因CLV1的基因组序列,多重比较后用以分析其开放阅读框、保守结构和单核苷酸多态性,用荧光实时定量PCR检测其在雄穗原基中的差异表达,并与雄穗分枝数和雄穗干重两个度量雄穗小的指标进行了相关分析。结果表明:KAP5-4基因的相对表达量与雄穗分枝数(r=0.77,P0.01)和雄穗干重正相关(r=0.83,P0.01)。11个自交系的CLV1基因开放框在2 104 bp存在单核苷酸多态性,其中5个自交系的2 014~2 016 bp核苷酸组成密码子GAC,编码受体样蛋白第702位酸性的天冬氨酸,另6个自交系的2 014~2 016 bp核苷酸组成密码子AAC,编码受体样蛋白第702位极性天冬氨酰胺。在前5个自交系中,CLV1基因的相对表达量与雄穗分枝数(r=-0.92,P0.01)和雄穗干重(r=-0.91,P0.05)负相关;而在后6个自交系中,仅与雄穗干重负相关(r=-0.91,P0.05)。综上所述,KAP5-4和CLV1基因的表达和单核苷酸多态性与玉米雄穗大小关系密切,可开发功能性的DNA标记用于玉米育种的分子标记辅助选择。     

Light induces jasmonate‐isoleucine conjugation via OsJAR1‐dependent and ‐independent pathways in rice

The bioactive form of jasmonate is the conjugate of the amino acid isoleucine (Ile) with jasmonic acid (JA), which is biosynthesized in a reaction catalysed by the GH3 enzyme JASMONATE RESISTANT 1 (JAR1). We examined the biochemical properties of OsJAR1 and its involvement in photomorphogenesis of rice (Oryza sativa). OsJAR1 has a similar substrate specificities as its orthologue in Arabidopsis. However, osjar1 loss‐of‐function mutants did not show as severe coleoptile phenotypes as the JA‐deficient mutants coleoptile photomorphogenesis 2 (cpm2) and hebiba, which develop long coleoptiles in all light qualities we examined. Analysis of hormonal contents in the young seedling stage revealed that osjar1 mutants are still able to synthesize JA‐Ile conjugate in response to blue light, suggesting that a redundantly active enzyme can conjugate JA and Ile in rice seedlings. A good candidate for this enzyme is OsJAR2, which was found to be able to catalyse the conjugation of JA with Ile as well as with some additional amino acids. In contrast, if plants in the vegetative stage were mechanically wounded, the content of JA‐Ile was severely reduced in osjar1, demonstrating that OsJAR1 is the most important JA‐Ile conjugating enzyme in the wounding response during the vegetative stage.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

Suppressor of sessile spikelets1 (Sosl): a dominant mutant affecting inflorescence development in maize

Suppressor of sessile spikeletsl (Sos1) is a dominant mutant of maize that blocks branching of the spikelet-pair primordium to form the sessile spikelet during ear development. As a result, Sos1 mutant ears and tassels possess single spikelets as opposed to the normal condition of paired spikelets, one sessile and the other pedicellate. Sos1 also causes a reduction in the number of tassel branches and the number of orthostichies (or cupule ranks) in the ear. The sos1 genetic locus maps to the short arm of maize chromosome 4. The Sos1 single spikelet phenotype appears similar to the single spikelet phenotype found in teosinte, the probable progenitor of maize. This similarity invites the hypothesis that sos1 had a role in the evolution of maize from teosinte. However, genetic mapping data and a comparison of the developmental basis of the single spikelet condition in the Sos1 mutant and teosinte demonstrate that their similar phenotypes result from distinct genetic-developmental mechanisms. These results indicate that sos1 was not involved in the evolution of maize and caution against drawing conclusions of homology based solely on similar adult phenotypes.     

Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.     

Maize host requirements for Ustilago maydis tumor induction

The biotrophic pathogen Ustilago maydis causes tumors by redirecting vegetative and floral development in maize (Zea mays L.). After fungal injection into immature tassels, tumors were found in all floral organs, with a progression of organ susceptibility that mirrors the sequential location of foci of cell division in developing spikelets. There is sharp demarcation between tumor-forming zones and areas with normal spikelet maturation and pollen shed; within and immediately adjacent to the tumor zone, developing anthers often emerge precociously and exhibit a range of developmental defects suggesting that U. maydis signals and host responses are restricted spatially. Male-sterile maize mutants with defects in anther cell division patterns and cell fate acquisition prior to meiosis formed normal adult leaf tumors, but failed to form anther tumors. Methyl jasmonate and brassinosteroid phenocopied these early-acting anther developmental mutants by generating sterile zones within tassels that never formed tumors. Although auxin, cytokinin, abscisic acid and gibberellin did not impede tassel development, the Dwarf8 mutant defective in gibberellin signaling lacked tassel tumors; the anther ear1 mutant reduced in gibberellin content formed normal tumors; and Knotted1, in which there is excessive growth of leaf tissue, formed much larger vegetative and tassel tumors. We propose the hypothesis that host growth potential and tissue identity modulate the ability of U. maydis to redirect differentiation and induce tumors.     

New insight in the Gibberellin biosynthesis and signal transduction

Gibberellin (GA) plays important roles through plant growth and development. However, where GA is synthesized inside a cell and how it regulates sex determination is obscure. We analyzed the classic dwarf1 (d1) mutant in maize and revealed that D1 encodes GA 3-oxidase converting inactive GA intermediates to bioactive GA. As such, the D1 protein marks the sites where GA is potentially synthesized. Interestingly, the D1 protein was found to localize in the cytosol and nucleus, a dual-localization coinciding with the GA receptor. The same result was found for GA 20-oxidase catalyzing the upstream reaction. These results suggest that GA can be synthesized in the cytosol and nucleus. The D1 protein was highly and specifically expressed in the stamen primordia in the ear florets, but low in the whole tassel. Hence it is possible that low level of GA in the tassel is insufficient to suppress stamen development. As jasmonic acid (JA) plays antagonistic role to GA in the tassel florets, here we propose a model to explain this antagonism effect on the regulation of the stamen and pistil organ development in the tassel florets in maize.     

Gene transfer to maize male reproductive structure by particle bombardment of tassel primordia

Summary Maize (Zea mays L.) tassel primordia were used as a target for particle bombardment, to assess the possibility of introducing foreign DNA into male reproductive structures. Transient expression of the -glucuronidase gene (GUS) or anthocyanin marker genes (C1 and B-Peru) driven by the CaMV 35S promoter was obtained in tassel primordia 24h after bombardment. Gold particles coated with DNA reached stamen primordia tissues, which eventually form the anthers and pollen. Bombarded tassels were also cultured in vitro and GUS activity was detected in the vascular tissue of mature anthers that developed within 4 weeks. This new approach represents a preliminary step toward pollen mediated transformation.