Correlation analysis of the transcriptome of growing leaves with mature leaf parameters in a maize RIL population

Background: To sustain the global requirements for food and renewable resources, unraveling the molecular networks underlying plant growth is becoming pivotal. Although several approaches to identify genes and networks involved in final organ size have been proven successful, our understanding remains fragmentary. Results: Here, we assessed variation in 103 lines of the Zea mays B73xH99 RIL population for a set of final leaf size and whole shoot traits at the seedling stage, complemented with measurements capturing growth dynamics, and cellular measurements. Most traits correlated well with the size of the division zone, implying that the molecular basis of final leaf size is already defined in dividing cells of growing leaves. Therefore, we searched for association between the transcriptional variation in dividing cells of the growing leaf and final leaf size and seedling biomass, allowing us to identify genes and processes correlated with the specific traits. A number of these genes have a known function in leaf development. Additionally, we illustrated that two independent mechanisms contribute to final leaf size, maximal growth rate and the duration of growth. Conclusions: Untangling complex traits such as leaf size by applying in-depth phenotyping allows us to define the relative contributions of the components and their mutual associations, facilitating dissection of the biological processes and regulatory networks underneath

F-Box protein FBX92 affects leaf size in Arabidopsis thaliana

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates

Combined large-scale phenotyping and transcriptomics in maize reveals a robust growth regulatory network

Leaves are vital organs for biomass and seed production because of their role in the generation of metabolic energy and organic compounds. A better understanding of the molecular networks underlying leaf development is crucial to sustain global requirements for food and renewable energy. Here, we combined transcriptome profiling of proliferative leaf tissue with indepth phenotyping of the fourth leaf at later stages of development in 197 recombinant inbred lines of two different maize (Zea mays) populations. Previously, correlation analysis in a classical biparental mapping population identified 1,740 genes correlated with at least one of 14 traits. Here, we extended these results with data from a multiparent advanced generation intercross population. As expected, the phenotypic variability was found to be larger in the latter population than in the biparental population, although general conclusions on the correlations among the traits are comparable. Data integration from the two diverse populations allowed us to identify a set of 226 genes that are robustly associated with diverse leaf traits. This set of genes is enriched for transcriptional regulators and genes involved in protein synthesis and cell wall metabolism. In order to investigate the molecular network context of the candidate gene set, we integrated our data with publicly available functional genomics data and identified a growth regulatory network of 185 genes. Our results illustrate the power of combining in-depth phenotyping with transcriptomics in mapping populations to dissect the genetic control of complex traits and present a set of candidate genes for use in biomass improvement

Genome fideltiy and DNA repair in plants: the characterization of the MBD4 DNA glycosylase homolog in Arabidopsis thaliana

The maintenance of genome stability is a continuous challenge for all organisms that is encountered by DNA repair mechanisms. In the human genome, the most important individual source of point mutations is spontaneous hydrolytic deamination of 5- methylcytosine (Sved and Bird, 1990; Krawczak et al., 1998; Fryxell and Moon, 2005). This results in the formation of G:T mismatches and, if not properly repaired, in C-to-T transitions after replication (Salser, 1977). To reduce the number of mutations generated by deamination of 5-methylcytosine, vertebrates have developed a pathway to specifically repair G:T mismatched base pairs to G:C (Neddermann and Jiricny, 1993; Hendrich et al., 1999). The first step in this pathway is the recognition of the mismatched base pair by a DNA glycosylase, MBD4 or TDG, followed by removal of the thymine and restoration of the abasic site by the BER pathway. In contrast to vertebrates, little is known about the impact of spontaneous hydrolytic deamination on genome stability in plants and about the capacity to repair the damage generated in this way in plants. To contribute to answering these questions, we aimed to establish the role of the homolog of the DNA glycosylase MBD4 in Arabidopsis thaliana, both in vitro and in vivo. Completion of the genome sequence of Arabidopsis thaliana allowed us to identify this homolog based on amino acid similarities with human, mouse and chicken MBD4 homologs (chapter 2). Also in poplar and rice, MBD4 homologs were identified, implying that this repair pathway is conserved in plants. In chapter 3, we describe our attempts to determine the biochemical activity of AtMBD4 in vitro, using different approaches, while chapter 4 focuses on the identification of interactors, to gain further insight into the biological function of AtMBD4. The temporal and spatial expression pattern of AtMBD4 is unravelled in chapter 5. Also, the effects of various environmental stimuli on AtMBD4 expression are analyzed. Chapter 6 reports on the development of a scorable system to monitor C-to-T transition mutations in Arabidopsis and the validation of the system. This system was then used to evaluate if C:G-to-T:A reversion frequencies were altered in AtMBD4 overexpressing and silenced plants. In addition, the response of these mutants to various genotoxic treatments was compared to wild type (chapter 7). G:T mismatches not only arise through deamination of 5-methylcytosine, but can also originate from replication errors. MSH2 is a protein involved in amongst others the repair of these replication errors. In chapter 8, the in vivo role of the Arabidopsis homolog AtMSH2 in the repair of G:T mismatches was determined using the scorable system described in chapter 6. Additionally, we analyzed the sensitivity of an AtMSH2::T-DNA insertion mutant to different genotoxic agents, aiming to further unravel the biological functions of AtMSH2

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 104 events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms

Development and Application of Novel Constructs to Score C:G-to-T:A Transitions and Homologous Recombination in Arabidopsis1[W]

We report on the development of five missense mutants and one recombination substrate of the β-glucuronidase (GUS)-encoding gene of Escherichia coli and their use for detecting mutation and recombination events in transgenic Arabidopsis (Arabidopsis thaliana) plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:G-to-T:A transitions in a symmetrical sequence context and are in that respect complementary to previously published GUS point mutants. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. We show that spontaneous mutation and recombination events took place. Mutagenic treatment of the plants with ethyl methanesulfonate and ultraviolet-C increased the number of mutations, validating the use of these constructs to measure mutation and recombination frequencies in plants exposed to biotic or abiotic stress conditions, or in response to different genetic backgrounds. Plants were also subjected to heavy metals, methyl jasmonate, salicylic acid, and heat stress, for which no effect could be seen. Together with an ethyl methanesulfonate mutation induction level much higher than previously described, the need is illustrated for many available scoring systems in parallel. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli

Additional file 1: Figure S1. of Correlation analysis of the transcriptome of growing leaves with mature leaf parameters in a maize RIL population

Mean minimum–maximum normalized values for all analyzed traits in parental lines and RILs. Fig. S2 Some examples of RILs and the parental lines at seedling stage, 27 days after sowing. Fig. S3 Coefficient of variation for the different traits measured. Fig. S4 PCA analysis of the phenotype data of the population. Fig. S5 Expression patterns of the top 1 % of genes (anti-)correlated with the different traits. Fig. S6 Overrepresented MapMan categories of top 1 % of genes (anti-)correlated with phenotypic traits. Fig. S7 Overrepresented MapMan categories of top 1 % of genes (anti-)correlated with final leaf size traits. (PDF 1976 kb