Characterization of a novel brachytic2 (br2) allele in maize (2016)

Starting from the Green Revolution era, cereals have been bred for dwarf or semi-dwarf phenotype for many advantages, such as increased allocation of energy to seed rather than stem, denser growth, increased resistance to storm damages and reduced loss during harvesting. \nMany dwarf mutants are characterized by alteration in hormones biosynthesis and signaling, as brassinosteroids (BR) and gibberellins (GA) pathways, while a defect in polar auxin transport (PAT) causes the sorghum mutant dwarf3 (dw3) and the maize brachytic2 (br2) mutations. Many br2 alleles have been isolated in maize. At Purdue University (Indiana USA), allelic tests conducted on the dwarf NC238 inbred line indicated that the NC238 dwarf phenotype is due to a new br2 allele. The isolation of a tall NC238 plant indicated the mutation is due to a transposon insertion that originated the tall revertant plant in a transposition event. Both dw3 and br2 are mutations affecting the gene orthologue of Arabidopsis thaliana ABCB1, which functions in auxin PAT. \nAuxin is a key regulator of plant growth: local biosynthesis, degradation and conjugation contribute to the modulation of IAA homeostasis at the cellular level. ABCBs, together with the PIN FORMED, the AUX1/LAX and the PILS auxin transporters, create a directional auxin flux from the site of synthesis - young tissues - to the site of action. Auxin transport, with local biosynthesis and conjugation, allows the establishment of an auxin gradient, and the local auxin maxima regulates many aspects of the embryonic development, stem cell maintenance, shoot architecture, and tropic growth responses, root development and gravitropism. To date, the majority of the studies on auxin PAT have been focused on Arabidopsis, while less is known in other species, such as maize. This thesis focuses on the characterization of br2:NC238 brachytic maize inbred line. In order to better characterize this maize mutant line, both a phenotypic and genotypic analysis of the NC238 dwarf plants were performed. In parallel, the brachytic 2 allele was introgressed in the B73 maize reference inbred line background. \nThe present thesis is composed of four chapters.The first chapter is an introduction on Maize biology and genetics and auxin synthesis, metabolism and roles. This section comprises the review I wrote when I was visiting scholar at Prof. Johal lab at Purdue University: "The role of auxin transporters in monocot development". The review describes the roles of auxin transporters in monocot development: maize, rice, sorghum and brachypodium, making comparison with Arabidopsis auxin carriers, when possible. Moreover, a section describing the plant transposable elements structure and classification is present.\nThe second chapter describes the morphological characteristics of NC238 line. It was previously reported alterations in stem vasculature in br2. Other paper described alteration in leaf cell size and leaf angle. Literature lacks a root system characterization. The develop stage when the br2:NC238 phenotype arise was identify. Seedlings' root system, and stem traits on field grown plants at anthesis (University of Padova farm "L.Toniolo") were measured. The internode length, node diameter, number of nodes, tassel branches and ear position along the stem were recorded. Moreover, 1m deep soil coring at the base of the stem were made, in order to study the root system profile. In addition the anatomy of the stem and auxin localization in stem tissues were observed.\nThe third chapter regards the genetic characterization of the mutation. Genomic clones and transcripts sequences from both br2:NC238 and the revertant plants were amplified and sequenced. A study on the nature of the br2:NC238 insertion was performed through analyses of the sequence and interrogation against genes, repeats and transposons databases. Moreover, expression analyses of auxin transporters encoding genes were performed. \nChapter four describes the genetic and phenotypic characterization of the br2:B73 line obtained by introgression of the br2:NC238 abcb1mutant allele in the B73 reference line. \nIn conclusion, br2:NC238 short phenotype is due to the shortening and thickness of the lower internodes. The other parts of the plant are normal. Phenotype appeared when plants were 7-leaf-old. br2:NC238 and the revertant plants were indistinguishable in the early stages of development. \nThe br2:NC238 mutation is caused by a insertion in a intronic region of abcb1 gene. The insertion is characterized by terminal Inverted repeats (TIRs) and Target Site Duplication (TSD) that are also present twice in the revert line. Despite the similarities with many known transposable elements, it was not possible to find the family the br2:NC238 insertion sequence belongs to. The insertion caused alteration in the gene transcription and alteration in the expression analyses of auxin transporters encoding genes at 3- and 7-leaf-old plant stem tissues were observed. Moreover, an increase in T02 Maize GDB predicted splicing form was observed.\nThe introgression of br2:NC238 abcb1 mutant allele in B73 reference line genome originated a short stature plant, br2 type. The result indicates the abcb1 mutant allele was responsible for the br2:NC238 phenotype and suggests the allele may be used in breeding programs to obtain lines with reduced height.\n