A FORWARD GENETICS APPROACH TO STUDY SEED AND SEEDLING DEVELOPMENT IN MAIZE.

Embryogenesis, germination and early phases of seedling growth represent critical phases in the plant life cycle and are probably the most important events in determining the success of an annual plant. A rapid and robust emergence positively influences the capacity of the plant to take advantage of the favourite environment and to compete with its neighbours. In the perspective of a more sustainable agriculture specific characters are envisaged for a crop seedling, such as the resistance to environmental critical abiotic as well as biotic factors. For these reasons key factors subtending plant developmental process and contributing to the achievement of a productive and robust plant have to be searched inside the genetic network that control embryo and seedling development. Among the different aspects affecting seedling development the two that will be analysed in this study play an important role also in the interplay with the environment. Hormones are endogenous signals governing seedling growth and architecture establishment but at the same time are able to induce plant response to environmental stress. Wax deposition is required for determining a correct embryo and seedling development, and provides, beside that, a protective barrier that plants produce in their early developmental phases to defend themselves from pathogens as well as from variation in environmental abiotic components, such as temperature and water availability. Here, we report the characterization of the mutants lilliputian 1-1 (lil1-1) and fused leaves 1-1 (fdl1-1), both ascribable to defective seedling (des) maize mutants. lilliputian 1-1 (lil1-1) is a monogenic recessive mutant of maize, isolated from an active Mutator (Mu) stock and attributed to the insertion of a Mutator1 element in the first exon of a the gene encoding the BR C-6 oxidase. The enzyme belongs to the superfamily of CYP85A proteins and catalyzes the final steps of brassinosteroid synthesis. lil1-1 mutant exhibits a reproducible phenotype consisting of a large primary root, extremely reduced stature and crinkly leaves. Recently, another dwarf mutant of maize impaired in the same brassinosteroid C-6 oxidase and showing a very similar phenotype of lil1-1 has been characterized and the corresponding gene was termed brasssinosteroid deficient 1 (brd1) Allelism between the two mutant alleles has been demonstrated in this work. Moreover, it has been observed that the exogenous application of brassinolide to the lil1 mutant seedlings resulted in a partial recovery of the lil1-1 phenotype. This observation is in agreement to what previously observed for brd1-m in maize and other Br-deficient mutants in Arabidopsis, rice and tomato. Differently from some of these mutants, i.e det2 of Arabidopsis, lil1 genotype does not influence the seed formation and development. It is evident that the comparison between homozygous lil1-1 mutant and Li11-1 wild-type seeds from the same segregating ear did not highlight any difference in weight. In addition, F2 progeny ears obtained from F1 heterozygous Lil11/lil1-1 or homozygous Lil1-1/Lil1-1 plants showed the same average kernel number and total kernel weight per ear and the average weight of single kernel. BRs are also involved in the modulation of stress responses. Water loss assays and measurement of gas exchange demonstrated that lil1-1 plants lost less water and maintained efficient gas exchange under drought stress for longer time than wild-type siblings. Our hypothesis is that lil1-1 mutant is more tolerant to drought stress because it is by default in a physiological water stress condition. A similar interpretation has been proposed to explain the behaviour of the det2 mutant in Arabidopsis that is deficient in a steroid reductase. The det2 mutant showed an enhanced resistance to general oxidative stress, correlated with a constitutive increase in superoxide dismutase (SOD) activity and increased transcript levels of the defence gene catalase (CAT). To confirm this hypothesis, other studies must be performed, among them the expression analysis of genes involved in dehydration stress. However, the hypothesis is at the moment supported by the observation that lil1-1 mutant plants show phenotypic traits that are generally present in plant subjected to water stress, i.e. inhibition of lateral root growth, reduction in leaf area and plant growth, enlarged leaf thickness and increased stomatal density. The fdl1-1 mutant, previously isolated in our laboratory, allowed the identification and functional analysis of a novel maize MYB gene. The fdl1-1 mutation was caused by an Enhancer/Suppressor (En/Spm) element insertion in the third exon of the sequence encoding ZmMYB94, a transcription factor of the R2R3-MYB subfamily. In this work, proof of gene identity was obtained using an RNAi approach and by the analysis of the mutant cDNA sequence. The first experiment ascertained the lesion in the third exon of the sequence encoding ZmMYB94. The second approach confirmed that the mutant transcript retains the En/Spm element. The fdl1-1 mutant phenotype is expressed at early stages of seedling development, from germination to the three-four leaves stage, causing a general delay in germination and seedling growth as well as phenotypic abnormalities. The main features of 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. A previous study showed that fusions could be attributable to the alterations in cuticle deposition and highlighted an irregular wax distribution on the mutant leaf surfaces. Phylogenetic analysis demonstrated that its closest Arabidopsis related genes, i.e. MYB30, MYB94 and MYB96 have all been implicated in the regulation of cuticular wax biosynthesis in Arabidopsis. To gain insight into the role exerted by ZmMYB94 a deeper characterization of cuticle components were therefore undertaken in this study by comparing mutant and wild-type tissues. We found a significant reduction of the amount of waxes in the mutant versus wild-type samples at earlier developmental stages. In particular, the production of C32 alcohols, which is the major compound of cuticular waxes in the maize seedling, resulted drastically reduced in the mutants and replaced by shorter chain alcohol (C26, C28 and C30) and alkane (C29). On this basis, we speculate that ZmMYB94 specifically affects the activity of enzymes involved in the elongation of long chain wax molecules at the C30—C32 step. In maize, some glossy mutants, i.e glossy 2 and glossy 4 show the same block in the long chain elongation. Thus, some of the subtending genes could be under the control of ZmMYB94. Contrary to fdl1-1, none of glossy mutant of maize so far characterized showed post-genital organ fusion. This difference could be due to a greater decrease (more than 90%) of epicuticular waxes observed in the fdl1-1 mutant than in glossy mutants. It is also conceivable that ZmMYB94 affects directly or indirectly the expression of a set of genes involved in the biosynthesis of very-long-fatty acids and the failure of multiple activities has caused a worsening of the phenotype. Alternatively, ZmMYB94 could regulate also some genes involved in the biosynthesis of other cutin components. Although only minor changes in the cutin load were observed in the fdl1-1 mutant, the affected components could be important for determining organ separation. Recent studies strongly support the idea that cuticular wax accumulation contributes to drought resistance. However, it is still not known in crops how wax related genes are regulated in response to drought. In our study, an increment of water loss in the mutant seedlings has been demonstrated and a correlation between the severity of the phenotype and the rate of water loss was revealed. Moreover, we found that the transcript level of ZmMYB94 increased in plant under drought stress condition. Similarly to AtMYB30, AtMYB94 and AtMYB96, which are considered positive regulators of wax biosynthesis during stress, it is conceivable that ZmMYB94 stimulates the activity of genes involved in cuticular waxes biosynthesis thus contributing to increase drought tolerance in the early phases of maize seedling growth. In conclusion, our study further indicate that the study of BR-related mutants and mutants impaired in cuticular waxes biosynthesis could be important for unravelling the molecular mechanisms underlying stress response in early developmental phases of cultivated plants and ultimately to identify new genetic tools of interest for their application in designing new breeding strategies.