Michela Landoni

A paramutation phenomenon is involved in the genetics of maize low phytic acid1-241 (lpa1-241) trait

So far, in maize, three classes of mutants involved in phytic acid biosynthesis have been isolated: lpa1, lpa2 and lpa3. In 2007, a gene tagging experiment performed by Shi et al. found that mutations in ZmMRP4 (multidrug resistance-associated proteins 4) gene cause lpa1 phenotype. In previous studies, we isolated and described a single recessive lpa mutation (originally named lpa241), which was allelic to the lpa1-1 mutant, and was consequently renamed lpa1-241. It showed a decrease in the expression of the myo-inositol (Ins)-3-phosphate synthase gene (mips1S). In this study, we present genetic and molecular analyses of the lpa1-241 mutation that indicate an epigenetic origin of this trait, that is, a paramutagenic interaction that results in meiotically heritable changes in ZmMRP4 gene expression, causing a strong pleiotropic effect on the whole plant. The use of a 5-Azacytidine treatment provided data suggesting an association between gene methylation and the lpa1-241 phenotype. To our knowledge, this is the first report of a paramutagenic activity not involving flavonoid biosynthesis in maize, but regarding a key enzyme of an important metabolic pathway in plants.

A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana

Calmodulin (CAM) is an ubiquitous calcium binding protein whose function is to translate the signals, perceived as calcium concentration variations, into the appropriate cellular responses. In Arabidopsisthaliana there are 4 CAM isoforms which are highly similar, encoded by 7 genes, and one possible explanation proposed for the evolutionary conservation of the CAM gene family is that the different genes have acquired different functions so that they play possibly overlapping but non-identical roles. Here we report the characterization of the Arabidopsis mutant cam2-2, identified among the lines of the gene-trapping collection EXOTIC because of a distorted segregation of kanamycin resistance. Phenotypic analysis showed that in normal growth conditions cam2-2 plants were indistinguishable from the wild type while genetic analysis showed a reduced transmission of the cam2-2 allele through the male gametophyte and in vitro pollen germination revealed a reduced level of germination in comparison with the wild type. These results provide genetic evidence of the involvement of a CAM gene in pollen germination and support the theory of functional diversification of the CAM gene family.

Characterization of the first dominant dwarf maize mutant carrying a single amino acid insertion in the VHYNP domain of the dwarf8 gene

The “green revolution” involving mainly wheat and rice was based on the use by breeders of semidominant mutations involved in the signal transduction pathway of Gibberellin (GA). In particular, mutations in the Reduced height (Rht) gene of wheat have been used to reduce plant height and consequently to avoid storm damage and lodging. These genes have been cloned and they encode for DELLA proteins which contain an N-terminal DELLA and a VHYNP domain essential for GA-dependent degradation of these proteins. In maize several mutations have been isolated which affect gibberellin biosynthesis and perception and in particular, mutations in Dwarf8 (D8) gene cause a severe dwarfing phenotype. D8 gene has been identified as an orthologue of Rht (Reduced height), Slr1(Slender rice 1) and Gibberellic Acid Insensitive (GAI) genes, this latter is a negative regulator of GA response in Arabidopsis. In this work, for the first time, we isolated and characterized a single amino acid insertion in the VHYNP domain of D8 maize gene causing the appearance of a dominant dwarf mutation. This spontaneous mutation, named D8-1023, showed a phenotype which is less severe in comparison with the other D8 mutants previously isolated which have modifications in the DELLA domain. This mutant appears to be an useful tool either to study the mechanism of GA-modulated growth in plants or to lower the height of maize tropical germplasm for breeding purposes.

A mutation in the FZL gene of Arabidopsis causing alteration in chloroplast morphology results in a lesion mimic phenotype

Lesion mimic mutants (LMMs) are a class of mutants in which hypersensitive cell death and defence responses are constitutively activated in the absence of pathogen attack. Various signalling molecules, such as salicylic acid (SA), reactive oxygen species (ROS), nitric oxide (NO), Ca2+, ethylene, and jasmonate, are involved in the regulation of multiple pathways controlling hypersensitive response (HR) activation, and LMMs are considered useful tools to understand the role played by the key elements of the HR cell death signalling cascade. Here the characterization of an Arabidopsis LMM lacking the function of the FZL gene is reported. This gene encodes a membrane-remodelling GTPase playing an essential role in the determination of thylakoid and chloroplast morphology. The mutant displayed alteration in chloroplast number, size, and shape, and the typical characteristics of an LMM, namely development of chlorotic lesions on rosette leaves and constitutive expression of genetic and biochemical markers associated with defence responses. The chloroplasts are a major source of ROS, and the characterization of this mutant suggests that their accumulation, triggered by damage to the chloroplast membranes, is a signal sufficient to start the HR signalling cascade, thus confirming the central role of the chloroplast in HR activation.

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355–368, 2000; Pilu et al. in Theor Appl Genet 107:980–987, 2003a; Shi et al. Nat Biotechnol 25:930–937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP6) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins’ transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.

Isolation and characterization of a new mutant allele of brachytic 2 maize gene

More than 40 monogenic dwarfing mutants have been described in maize; however, the majority of these lead to great reductions in grain yield and, consequently, they have not been used to enhance crop yield in germplasm that is sensible to lodging. An exception in terms of commercial value is the maize mutant brachytic 2 (br2). Br2 gene was cloned in 2003, and it encodes for a protein most probably involved in auxin polar transport. We have isolated a new brachytic mutation that is allelic to the br2 locus and denoted this novel mutant as br2–23. Characterization of this mutant revealed that the br2 mutation modified not only the length of the internodes, as previously reported, but the structure of the leaves as well. Br2–23/br2–23 heterozygotes have a useful intermediate phenotype in terms of plant height, ear height and leaf angle, suggesting a possible utilization of this effect in developing new hybrids. This mutant also appears to be an useful tool by which to study the switch points of the complex developmental program determining maize plant height and architecture.

Low Phytic Acid 1 Mutation in Maize Modifies Density, Starch Properties, Cations, and Fiber Contents in the Seed

Monogastric animals are unable to digest phytic acid, so it represents an antinutritional factor and also an environmental problem. One strategy to solve this problem is the utilization of low phytic acid (lpa) mutants that accumulate low levels of phytic P and high levels of free phosphate in the seeds; among the lpa maize mutants lpa1 exhibited the highest reduction of phytic acid in the seed. This study indicated that the low phytic acid mutations exerted pleiotropic effects not directly connected to the phytic acid pathway, such as on seed density, content of ions, and the antioxidant compounds present in the kernels. Furthermore some nutritional properties of the flour were altered by the lpa1 mutations, in particular lignin and protein content, while the starch does not seem to be modified as to the total amount and in the amylose/amylopectin ratio, but alterations were noticed in the structure and size of granules.

Analysis of chromosome number and speculations on the origin of Arundo donax L. (Giant Reed)

Arundo donax (commonly called Giant Reed) is a perennial rhizomatous grass native to Asia, nowadays diffused all over the world. Due to its high biomass production and great adaptability to marginal land, interest in this species is increasing. In fact A. donax could represent an important and promising energy crop for heat and bioethanol second generation production. The propagation of A. donax is strictly agamic by rhizome fragmentation and cane node germination, strongly limiting the possibility of genetic improvement by breeding. The sterility could be caused by the fact that A. donax is a hybrid with uneven ploidy or a triploid species. It is difficult to propose an explanation for its sterility, because the chromosome number of A. donax is still a matter of debate, due to the high number and small size of the chromosomes; in the bibliography different counts ranging from 40 to 110 are reported. With the aim of establishing the chromosome number of A. donax we selected and counted 17 metaphase plates prepared from root tips obtained by hydroponic cultivation of cane nodes; our counts showed that A. donax most probably has 110 chromosomes. Our results suggested us two possible hypotheses, also based on SSR molecular marker results, concerning the evolutionary processes involved in the origins of A. Donax.

Characterization of the Ra1 maize gene involved in inflorescence architecture

The genetic and molecular control of inflorescence and flower development has been extensively studied in model dicotyledonous plants such as Arabidopsis but even now little is known about monocotyledonous species. In maize several mutants have been isolated that perturbed normal inflorescence development. In particular, the Ramosa1 (Ra1) gene, coding for a zinc finger transcription factor, plays a role in inflorescence structure by determining the number of branches. Although the mechanism by which Ra1 acts is unclear, inflorescence meristems in these regions assume a branch meristem identity rather than becoming spikelet pairs. In this work we characterize a new mutation of Ra1 gene that originated spontaneously from a B73 inbred line. This loss-of-function mutation is caused by the deletion of the lysine residue at position 53 in the RA1 putative zinc-finger domain. This is the first evidence for a single amino acid deletion in the zinc finger domain that knocks out the function of the RA1 protein. This result strongly suggests that the RA1 protein functions by acting as a DNA-binding protein, probably involved in transcriptional regulation. Furthermore, Ra1 overexpression in the Arabidopsis ortholog superman (sup) mutant, whose flowers are characterized by the presence of additional stamens, was not able to restore the correct stamen number, indicating that SUP and Ra1 genes do not share an identical function.

The brachytic 2 and 3 maize double mutant shows alterations in plant growth and embryo development

In maize there are three types of brachytic mutants (br1, br2 and br3) showing short stature and a gibberellins-insensitive phenotype. So far only the brachytic 2 gene has been cloned and it encodes for a putative protein of the Multidrug Resistant (MDR) class of P-glycoproteins (PGPs) that could be involved in polar movement of auxins: in fact the br2 mutant is insensitive to treatment with auxins and gibberellins. We have isolated a new recessive mutation of br2 gene (named br2-23) and with the aim of study its interactions with the other brachytic mutations we produced a br2 br3 double mutant that showed an additive effect on the stature with respect to the single mutants br2 and br3 and abnormal growth. In the progeny of the selfed double mutant we observed various defective seedlings, mirroring an altered embryo development and growth, which also suggested a role for the br3 gene in auxin transport. Expression analysis of the auxin efflux transporters codified by ZmPIN1 genes supports this finding, showing the up-regulation of the ZmPIN1a gene in the br3 mutant. To our knowledge this is the first report showing the involvement of Br2 and Br3 genes in embryo development. These single and double mutants appear to be useful tools to study the genetics of plant height and to investigate auxin transport in plants.