FUNCTIONAL ANALYSIS OF MAIZE GENES INVOLVED IN SEEDLING DEVELOPMENT AND IN PLANT ¿ ENVIRONMENT INTERACTION

Maize is one of the most important cultivated plants on a world scale, with a total production which exceeds one billion tonnes per year (FAOSTAT 2017). It is an important human food source in many parts of the world, but is also intensely exploited for biofuel and feed production. Furthermore, maize is also a model plant for research studies in the field of biology and genetics. For these reasons, maize is one of the most intensively studied crops, with the aim of improving its productivity, which in the USA has increased nearly five times since the 1940s. This dramatic yield improvement is due to the development and widespread use of new farming technologies. An important role was played by genetic improvement, with the use of highly productive maize hybrids and, more recently, biotechnology. However, the increases in annual productivity of maize and the other main crops exploited for food production seem to have reached a stall phase in recent years. It has been estimated that the world population will increase from 7.5 to 9.7 billion people by 2050. To meet the food needs of the increasing population, and to satisfy diets that will include more meat, according to the Food and Agricultural Organization (FAO), worldwide crop production will have to increase by 70%. It is a tough task, made even more difficult by the fact that the worldwide area of cultivable soil is decreasing as a consequence of increasing urbanization and climate change. To reach the target, new strategies are required, which will include multiple and integrated approaches, among them genetic improvement. One of the main challenges will be to develop new plant ideotypes that will combine the capability to tolerate biotic and abiotic stress with no reduction in yield. Within this perspective, the work carried out in this thesis project was aimed at understanding the molecular mechanisms involved in plant response to environmental factors. The work was organized in two parts, which are presented here in two different chapters. Both chapters are focused on genes that control plant development as well as plant- environment interactions. The first chapter deals with the study of the genetic regulation of cuticle deposition in maize. The cuticle is an important plant organ and constitutes the first barrier against many environmental stresses, including water deprivation and pathogen interaction. The cuticle is produced by epidermal cells and is composed of a complex array of long chain hydrocarbons, constantly deposed on the aerial surface for all the plant’s life. To investigate the cuticle biosynthesis process in maize, our strategy was based on the functional characterization of ZmMYB94, also known as fused leaves 1 (fdl1). This gene encodes a transcription factor of the R2R3-MYB subfamily, expressed in embryo, seedling and silk tissues. A mutant in this gene, referred to as fdl1-1, was available for this study. It originated from the insertion of an Enhancer/Suppressor (En/Spm) element in the third exon of the ZmMYB94 gene. The mutation has a pleiotropic effect on seedling development. The main features of fdl1-1 mutant plants are irregular coleoptile opening and the presence of regions of adhesion between the coleoptile and the first leaf and between the first and second leaves. Deeper studies of the fdl1-1 mutant, performed by electron microscopy analysis, showed that, in regions of organ adhesion, cuticle was absent and, on the epidermal surface, epicuticular wax deposition occurred irregularly. These observations led to the hypothesis that phenotypic alterations observed in the mutant seedlings may be attributable to defects in the cuticle-related biosynthetic pathways. To gain insight into the role of fdl1 in controlling cuticle formation, a large-scale RNA sequencing analysis was carried out in this work. By means of this approach, more than one thousand differentially expressed genes (DEGs) were found in the fdl1-1 mutant compared with the wild type seedling transcriptome. The analysis of single DEGs confirmed that fdl1 is involved in the regulation of the biosynthesis of both cuticle components, since genes for the cuticular waxes deposition as well as cutin biosynthesis were detected. In particular, five genes with a reduced expression level and one with an increased expression level in the fdl1-1 mutant encoded for enzymes involved in the Fatty acid elongation complex, the main source of basic compounds exploited in cutin and waxes biosynthesis. Furthermore, two genes found with a reduced expression level in the mutant were also involved in the cutin biosynthesis. Interesting sidelights on these two genes are available from the study of their orthologues in rice (OsONI3) and in Arabidopsis (AtEDA17). The knock-out mutant of ONI3 in rice shows a phenotype that is very similar to that of the fdl1-1 mutant in maize, with defects in cutin deposition and fusions between the coleoptile and the first leaves. Moreover, the knock-out mutant of EDA17 in Arabidopsis shows defects in cutin deposition and fusions between floral organs. Other two genes found differentially expressed in fdl1-1 encode for enzymes involved in epicuticular waxes biosynthesis, thus explaining the defects observed in fdl1-1 mutants. On the basis of these observations, we developed a model that explains the mode of action of fdl1 in controlling cuticle formation. Furthermore, the large-scale RNA sequencing analysis done in this study revealed a considerable number of genes differentially expressed in the fdl1-1 mutant that are involved in other important processes, such as plant clock regulation, plant pathogen interaction and hormone signalling. A deeper investigation of the role of these genes will be of help in elucidating the action of fdl1 in controlling different aspects of plant development. We also demonstrated in the present study that fdl1 is actively involved in the drought stress response in maize seedlings. Indeed, an expression analysis showed strong differences in the fdl1 transcript level in seedlings exposed to drought stress condition compared with seedlings grown in well-watered conditions. Indeed, in normally watered plants, the fdl1 transcript level constantly increased during leaf expansion, until it reached a high expression level. In plants exposed to drought stress conditions, the fdl1 transcript level showed a different pattern, with a strong increment in the first day of stress, and then a decrease until the initial level was reached and maintained for the whole duration of drought conditions. We may speculate that the involvement of fdl1 in the plant response to drought conditions, consists in promoting a modification of cuticle composition that will reduce water loss. To further investigate the involvement of fdl1 in drought stress tolerance, its orthologous gene was identified in Eragrostis curvula. This species is particularly interesting for our purposes, because its genome is very similar to the maize genome. Furthermore, unlike maize, in Eragrostis different ecotypes, characterized by different drought stress tolerance, have been described. In a preliminary analysis, we observed that the expression pattern of Ecfdl1 is different among three Eragrostis ecotypes. Differently from what was observed in maize, Ecfdl1 appeared to be expressed in different adult tissues. Moreover, differences in the Ecfdl1 expression profile were detected in different ecotypes. Besides in young maize tissues, fdl1 also appears to have an active role in controlling cuticle deposition in silk tissues. We detected differences between the fdl1-1 mutant and wild type plants in the composition of the wax layer covering the silks. Since maize silks constitute the main route of entry for pathogens to reach the seeds, in our opinion the differences found in silk coverage can influence the plant-pathogen interaction. This hypothesis was confirmed by comparing fusarium ear rot symptoms between wild type and fdl1-1 mutant ears experimentally inoculated with Fusarium verticillioides in a two year experiment. Our results seem to indicate that the fdl1-1 mutant is less susceptible to fusarium infection compared with the wild type. In conclusion to this part of the work, our studies provided details about the involvement of fdl1 in the regulation of cuticle biosynthesis and deposition during two of the most important moments of the plant life cycle: the seedling stage and silk development. Furthermore, in these two delicate moments, we have evidence showing that fdl1 plays an active role in drought stress response and pathogen interaction. In the second chapter, the role of brassinosteroids (BRs) in leaf permeability and architecture was further analysed. BRs are a class of steroid hormones essential for plant growth and development. BRs are involved in many developmental traits of agronomic importance such as seed germination, plant architecture, flowering time and seed yield. In addition to having an important role in development, brassinosteroids exert anti-stress effects on plants and are essential for the ability of plants to adapt to abiotic stresses. This part of the work was focused on the characterization of the maize lilliputian1-1 (lil1-1) mutant, which is impaired in one of the last steps of brassinosteroid biosynthesis. The subtending gene putatively encodes for a brassinosteroid C-6 oxidase (brC-6 oxidase). The mutant appears severely compromised in height, floral development and overall plant architecture. Leaf primordia are more compressed compared with wild type, and mutant leaves appear thicker than wild type leaves, exhibiting altered shape and the presence of supernumerary cell layers in the mesophyll region between the leaf vessels and the adaxial leaf epidermis. In this study, alterations in epicuticular waxes deposition were found in the lil1-1 mutant. Furthermore, it was shown that the leaf epidermis of lil1-1 shows a significantly lower permeability than wild type. These findings are in accordance with previous observations obtained in our laboratory, which showed that the lil1-1 mutant shows a better dehydration tolerance. In our hypothesis, the thicker epidermis observed in lil1-1 compared with wild type, can explain the lower permeability and the better dehydration tolerance. Furthermore, in this chapter we used the lil1-1 mutant to better investigate the BRs biosynthesis pathway. This is a complex pathway and, although it has been the subject of several studies in recent years, some aspects are still to be clarified. For this purpose, we analysed the interaction between the lil1-1 mutant, and another well-known maize BRs mutant, i.e. the nana1-1 (na1-1) mutant. Both nana1 and lil1 genes have key roles in the BR biosynthesis pathway. The product of nana1 is involved in two parallel pathways, therefore lack of its action may lead to an interruption of both. The lil1 gene product, however, is involved in the last steps of the pathway, leading to the formation of castasterone and brassinolide. Our analysis revealed that lil1-1 is epistatic to na1-1. These data suggest the existence in the maize BR pathway of an additional na1-independent branch leading to the production of CS precursors. In conclusion, this part of the work demonstrated the involvement of brassinosteroids in passive leaf permeability and provided new information that will be useful to unravel the complex BR biosynthetic pathway in plants. Overall, the work developed in this thesis project provides indications useful to better understanding the genetic mechanisms that regulate plant resistance to drought and pathogens. A good comprehension of these mechanisms can ultimately be useful to identify new genetic tools of interest, and to develop crops more adapted to the challenges of the future. Because of the appointment with 2050, only 32 annual production cycles remain.