Tnt1 retrotransposon tagging of STF in Medicago truncatula reveals tight coordination of metabolic, hormonal and developmental signals during leaf morphogenesis

Tnt1 (transposable element if Nicotiana tabaccum cell type 1) is one of the very few active LTR retrotransposons used for gene tagging in plants. In the model legume Medicago truncatula, Tnt1 has been effectively used as a gene knock-out tool to generate several very useful mutants. stenofolia (stf) is such a mutant identified by Tnt1 insertion in a WUSCHEL-like homeobox transcription factor. STF is required for blade outgrowth, leaf vascular patterning and female reproductive organ development in barrel medic and woodland tobacco. Using transcript profiling and metabolite analysis, we uncovered that mutant leaves are compromised in steady-state levels of multiple phytohormones, sugar metabolites and derivatives including flavonoids and polyamines. In the lam1 mutant (caused by deletion of the STF ortholog in Nicotiana sylvestris), while glucose, fructose, mannose, galactose, myo-inositol and aromatic aminoacids are dramatically reduced, sucrose is comparable to wild-type levels, and glutamine, proline, putrescine, nicotine and sorbitol are highly increased. We demonstrated that both stf and lam1 mutants accumulate reduced levels of free auxin and ABA in their leaves, and ectopic expression of STF in tobacco leads to auxin and cytokinin overproduction phenotypes including formation of tumors on the roots and crown. These data suggest that STF mediated integration of metabolic and hormonal signals are required for lateral organ morphogenesis and elaboration

Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens

Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR), which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS)-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance

An efficient and improved method for virus-induced gene silencing in sorghum

Background: Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. Methods: Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. Results: This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. Conclusion: In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum

Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens

Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised in nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens

Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested

Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction During Nonhost Resistance in Nicotiana benthamiana and Arabidopsis

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H2O2 accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response–causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H2O2 during both gene-for-gene and nonhost resistance responses

GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata

Formate Dehydrogenase (FDH1) Localizes to Both Mitochondria and Chloroplast to Play a Role in Host and Nonhost Disease Resistance

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In this study, the metabolic enzyme formate dehydrogenase (FDH1) was identified to be involved in nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. Arabidopsis Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that SA is most likely involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to only mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 localized to mitochondria and the amount of FDH1 localized to mitochondria increased upon infection with host or nonhost pathogens. Interestingly, the subcellular localization of FDH1 was observed in both mitochondria and chloroplasts after infection with a nonhost pathogen in Arabidopsis. We speculate that FDH1 plays a role in cellular signaling networks between mitochondria and chloroplasts to produce coordinated defense responses such as SA-induced reactive oxygen species (ROS) generation and hypersensitive response (HR)-induced cell death against nonhost bacterial pathogens

Functional specialization of duplicated AP3-like genes in Medicago truncatula

This is the accepted version of the following article: Roque, E., Serwatowska, J., Cruz Rochina, M., Wen, J., Mysore, K. S., Yenush, L., Beltrán, J. P. and Cañas, L. A. (2013), Functional specialization of duplicated AP3-like genes in Medicago truncatula. Plant J, 73: 663–675 , which has been published in final form at http://dx.doi.org/10.1111/tpj.12068The Bclass of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M.truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.This work was funded by grants BIO2006-09374 and BIO2009-08134 from the Spanish Ministry of Science and Innovation. We are gratefully to Mario A. Fares and Santiago F. Elena (Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for helpful comments and bioinformatics support. The collaboration and assistance of Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged.Roque Mesa, EM.; Serwatowska, J.; Rochina Peñalver, MC.; Wen, J.; Mysore, KS.; Yenush, L.; Beltran Porter, JP.... (2013). Functional specialization of duplicated AP3-like genes in Medicago truncatula. The Plant Journal. 73(4):663-675. doi:10.1111/tpj.12068S663675734Altschul, S. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402. doi:10.1093/nar/25.17.3389Aoki, S., Uehara, K., Imafuku, M., Hasebe, M., & Ito, M. (2004). Phylogeny and divergence of basal angiosperms inferred from APETALA3- and PISTILLATA-like MADS-box genes. Journal of Plant Research, 117(3). doi:10.1007/s10265-004-0153-7Baum, D. (2002). Response: Missing links: the genetic architecture of flower and floral diversification. Trends in Plant Science, 7(1), 31-34. doi:10.1016/s1360-1385(01)02181-1A., B., K., K., A., F., C., V., M.-A., L., H., S., & G., T. (2002). A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes. Molecular Genetics and Genomics, 266(6), 942-950. doi:10.1007/s00438-001-0615-8Benlloch, R., d’ Erfurth, I., Ferrandiz, C., Cosson, V., Beltrán, J. P., Cañas, L. A., … Ratet, P. (2006). Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes. Plant Physiology, 142(3), 972-983. doi:10.1104/pp.106.083543Benlloch, R., Roque, E., Ferrándiz, C., Cosson, V., Caballero, T., Penmetsa, R. V., … Madueño, F. (2009). Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development inMedicago truncatula. The Plant Journal, 60(1), 102-111. doi:10.1111/j.1365-313x.2009.03939.xBerbel, A., Navarro, C., Ferrándiz, C., Cañas, L. A., Beltrán, J.-P., & Madueño, F. (2005). Functional Conservation of PISTILLATA Activity in a Pea Homolog Lacking the PI Motif. Plant Physiology, 139(1), 174-185. doi:10.1104/pp.104.057687Bowman, J. L., Smyth, D. R., & Meyerowitz, E. M. (1989). Genes directing flower development in Arabidopsis. The Plant Cell, 1(1), 37-52. doi:10.1105/tpc.1.1.37Broholm, S. K., Pöllänen, E., Ruokolainen, S., Tähtiharju, S., Kotilainen, M., Albert, V. A., … Teeri, T. H. (2009). Functional characterization of B class MADS-box transcription factors in Gerbera hybrida. Journal of Experimental Botany, 61(1), 75-85. doi:10.1093/jxb/erp279Cheng, X., Wen, J., Tadege, M., Ratet, P., & Mysore, K. S. (2010). Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants. Plant Reverse Genetics, 179-190. doi:10.1007/978-1-60761-682-5_13Coen, E. S., & Meyerowitz, E. M. (1991). The war of the whorls: genetic interactions controlling flower development. Nature, 353(6339), 31-37. doi:10.1038/353031a0Coronado, C., Zuanazzi, J., Sallaud, C., Quirion, J. C., Esnault, R., Husson, H. P., … Ratet, P. (1995). Alfalfa Root Flavonoid Production Is Nitrogen Regulated. Plant Physiology, 108(2), 533-542. doi:10.1104/pp.108.2.533Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1(4), 19-21. doi:10.1007/bf02712670Drea, S., Hileman, L. C., de Martino, G., & Irish, V. F. (2007). Functional analyses of genetic pathways controlling petal specification in poppy. Development, 134(23), 4157-4166. doi:10.1242/dev.013136D’ Erfurth, I., Cosson, V., Eschstruth, A., Lucas, H., Kondorosi, A., & Ratet, P. (2003). Efficient transposition of theTnt1tobacco retrotransposon in the model legumeMedicago truncatula. The Plant Journal, 34(1), 95-106. doi:10.1046/j.1365-313x.2003.01701.xFerr�ndiz, C., Navarro, C., G�mez, M. D., Ca�as, L. A., & Beltr�n, J. P. (1999). Flower development inPisum sativum: From the war of the whorls to the battle of the common primordia. Developmental Genetics, 25(3), 280-290. doi:10.1002/(sici)1520-6408(1999)25:33.0.co;2-3Geuten, K., & Irish, V. (2010). Hidden Variability of Floral Homeotic B Genes in Solanaceae Provides a Molecular Basis for the Evolution of Novel Functions. The Plant Cell, 22(8), 2562-2578. doi:10.1105/tpc.110.076026Goto, K., & Meyerowitz, E. M. (1994). Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes & Development, 8(13), 1548-1560. doi:10.1101/gad.8.13.1548Heard, J., & Dunn, K. (1995). Symbiotic induction of a MADS-box gene during development of alfalfa root nodules. Proceedings of the National Academy of Sciences, 92(12), 5273-5277. doi:10.1073/pnas.92.12.5273Hecht, V., Foucher, F., Ferrándiz, C., Macknight, R., Navarro, C., Morin, J., … Weller, J. L. (2005). Conservation of Arabidopsis Flowering Genes in Model Legumes. Plant Physiology, 137(4), 1420-1434. doi:10.1104/pp.104.057018The evolution of functionally novel proteins after gene duplication. (1994). Proceedings of the Royal Society of London. Series B: Biological Sciences, 256(1346), 119-124. doi:10.1098/rspb.1994.0058Irish, V. F. (2006). Duplication, Diversification, and Comparative Genetics of Angiosperm MADS‐Box Genes. Advances in Botanical Research, 129-161. doi:10.1016/s0065-2296(06)44003-9Jack, T., Fox, G. L., & Meyerowitz, E. M. (1994). Arabidopsis homeotic gene APETALA3 ectopic expression: Transcriptional and posttranscriptional regulation determine floral organ identity. Cell, 76(4), 703-716. doi:10.1016/0092-8674(94)90509-6Kim, S., Yoo, M.-J., Albert, V. A., Farris, J. S., Soltis, P. S., & Soltis, D. E. (2004). Phylogeny and diversification of B-function MADS-box genes in angiosperms: evolutionary and functional implications of a 260-million-year-old duplication. American Journal of Botany, 91(12), 2102-2118. doi:10.3732/ajb.91.12.2102Kramer, E. M., & Irish, V. F. (2000). Evolution of the Petal and Stamen Developmental Programs: Evidence from Comparative Studies of the Lower Eudicots and Basal Angiosperms. International Journal of Plant Sciences, 161(S6), S29-S40. doi:10.1086/317576Kramer, E. M., Di Stilio, V. S., & Schlüter, P. M. (2003). Complex Patterns of Gene Duplication in the APETALA3 and PISTILLATA Lineages of the Ranunculaceae. International Journal of Plant Sciences, 164(1), 1-11. doi:10.1086/344694Kramer, E. M., Su, H.-J., Wu, C.-C., & Hu, J.-M. (2006). BMC Evolutionary Biology, 6(1), 30. doi:10.1186/1471-2148-6-30Lamb, R. S., & Irish, V. F. (2003). Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages. Proceedings of the National Academy of Sciences, 100(11), 6558-6563. doi:10.1073/pnas.0631708100Liu, Y., Nakayama, N., Schiff, M., Litt, A., Irish, V. F., & Dinesh-Kumar, S. P. (2004). Virus Induced Gene Silencing of a DEFICIENS Ortholog in Nicotiana Benthamiana. Plant Molecular Biology, 54(5), 701-711. doi:10.1023/b:plan.0000040899.53378.83De Martino, G., Pan, I., Emmanuel, E., Levy, A., & Irish, V. F. (2006). Functional Analyses of Two Tomato APETALA3 Genes Demonstrate Diversification in Their Roles in Regulating Floral Development. The Plant Cell, 18(8), 1833-1845. doi:10.1105/tpc.106.042978Ohno, S. (1970). Evolution by Gene Duplication. doi:10.1007/978-3-642-86659-3Páez-Valencia, J., Sánchez-Gómez, C., Valencia-Mayoral, P., Contreras-Ramos, A., Hernández-Lucas, I., Orozco-Segovia, A., & Gamboa-deBuen, A. (2008). Localization of the MADS domain transcriptional factor NMH7 during seed, seedling and nodule development of Medicago sativa. Plant Science, 175(4), 596-603. doi:10.1016/j.plantsci.2008.06.008Pnueli, L., Abu-Abeid, M., Zamir, D., Nacken, W., Schwarz-Sommer, Z., & Lifschitz, E. (1991). The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes fromAntirrhinumandArabidopsis. The Plant Journal, 1(2), 255-266. doi:10.1111/j.1365-313x.1991.00255.xRiechmann, J. L., Krizek, B. A., & Meyerowitz, E. M. (1996). Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proceedings of the National Academy of Sciences, 93(10), 4793-4798. doi:10.1073/pnas.93.10.4793Rijpkema, A. S., Royaert, S., Zethof, J., van der Weerden, G., Gerats, T., & Vandenbussche, M. (2006). Analysis of the Petunia TM6 MADS Box Gene Reveals Functional Divergence within the DEF/AP3 Lineage. The Plant Cell, 18(8), 1819-1832. doi:10.1105/tpc.106.042937Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P. J., Hansen, R., Tetens, F., … Sommer, H. (1992). Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. The EMBO Journal, 11(1), 251-263. doi:10.1002/j.1460-2075.1992.tb05048.xSoltis, P. S., Brockington, S. F., Yoo, M.-J., Piedrahita, A., Latvis, M., Moore, M. J., … Soltis, D. E. (2009). Floral variation and floral genetics in basal angiosperms. American Journal of Botany, 96(1), 110-128. doi:10.3732/ajb.0800182Sommer, H., Beltrán, J. P., Huijser, P., Pape, H., Lönnig, W. E., Saedler, H., & Schwarz-Sommer, Z. (1990). Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors. The EMBO Journal, 9(3), 605-613. doi:10.1002/j.1460-2075.1990.tb08152.xStellari, G. M., Jaramillo, M. A., & Kramer, E. M. (2004). Evolution of the APETALA3 and PISTILLATA Lineages of MADS-Box–Containing Genes in the Basal Angiosperms. Molecular Biology and Evolution, 21(3), 506-519. doi:10.1093/molbev/msh044Tadege, M., Ratet, P., & Mysore, K. S. (2005). Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula. Trends in Plant Science, 10(5), 229-235. doi:10.1016/j.tplants.2005.03.009Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., … Mysore, K. S. (2008). Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. The Plant Journal, 54(2), 335-347. doi:10.1111/j.1365-313x.2008.03418.xTamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution, 24(8), 1596-1599. doi:10.1093/molbev/msm092Taylor, S., Hofer, J., & Murfet, I. (2001). Stamina pistilloida, the Pea Ortholog of Fim and UFO, Is Required for Normal Development of Flowers, Inflorescences, and Leaves. The Plant Cell, 13(1), 31-46. doi:10.1105/tpc.13.1.31Theissen, G., & Melzer, R. (2007). Molecular Mechanisms Underlying Origin and Diversification of the Angiosperm Flower. Annals of Botany, 100(3), 603-619. doi:10.1093/aob/mcm143Tröbner, W., Ramirez, L., Motte, P., Hue, I., Huijser, P., Lönnig, W. E., … Schwarz-Sommer, Z. (1992). GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. The EMBO Journal, 11(13), 4693-4704. doi:10.1002/j.1460-2075.1992.tb05574.xTucker, S. C. (2003). Floral Development in Legumes. Plant Physiology, 131(3), 911-926. doi:10.1104/pp.102.017459Urbanus, S. L., de Folter, S., Shchennikova, A. V., Kaufmann, K., Immink, R. G., & Angenent, G. C. (2009). In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana. BMC Plant Biology, 9(1), 5. doi:10.1186/1471-2229-9-5Vandenbussche, M., Zethof, J., Royaert, S., Weterings, K., & Gerats, T. (2004). The Duplicated B-Class Heterodimer Model: Whorl-Specific Effects and Complex Genetic Interactions in Petunia hybrida Flower Development. The Plant Cell, 16(3), 741-754. doi:10.1105/tpc.019166Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., … Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581-590. doi:10.1046/j.1365-313x.2001.01105.xWu, C., Ma, Q., Yam, K.-M., Cheung, M.-Y., Xu, Y., Han, T., … Chong, K. (2005). In situ expression of the GmNMH7 gene is photoperiod-dependent in a unique soybean (Glycine max [L.] Merr.) flowering reversion system. Planta, 223(4), 725-735. doi:10.1007/s00425-005-0130-