A maize cell wall database

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- Why is a cell wall gene catalogue and macroarray useful in maize ?

- What does MAIZEWALL contain ?

- How was MAIZEWALL built ?

- Sample description

- Cell wall macroarray construction and hybridization

Why is a cell wall gene catalogue and macroarray useful in maize ?

The plant cell wall provides structural support and defines cell size and shape, thereby playing a critical role in determining cell function. Primary cell walls are laid down in all plant cells and are mainly composed of cellulose, hemicellulose, pectin and protein. The structure and composition differ greatly among species, and especially between dicots and monocots. Dicots and non-commelinoid monocots possess type I cell walls, whereas type II cell walls are found in commelinoid monocotyledons which include maize and rice. For example, the primary wall in maize contains less pectins and proteins and significant amounts of hydroxycinnamic acids. During the differentiation of specialized cell types such as fibers and xylem tracheary elements (TEs), in addition to primary walls, secondary walls are subsequently deposited to insure additional mechanical strength and solute conduction. Although much information is available for specific genes involved in dicot cell wall formation, nearly nothing is known in monocots, and in particular in maize. For this reason, we constructed a centralized cell wall database in maize that could be extremely useful for the scientific community working on cell walls and/or maize.
Genes involved in cell wall biosynthesis and assembly are often encoded by multigene families. This creates an added complexity in assigning a precise role of a given gene family member to a given biological function. For this reason, a gene-specific-tag for each gene was spotted on the maize cell wall macroarray. Not only does the macroarray insured gene-specific expression, but it also provides a tool to perform large numbers of hybridizations that are less labor intensive and less costly than microarrays, and that are adapted to resolve questions specifically related to cell wall metabolism in maize.
And finally, from an applied perspective, the plant cell wall is a determinant of forage quality. Among forage plants, silage maize is a major food crop for cattle feeding during winter and summer when grazing is not possible. The availability of genetic and genomic tools are currently becoming available in maize, and beyond its agronomic interest, make it a graminaceous model plant to study cell wall formation, lignification and digestibility.

What does MAIZEWALL contain ?

MAIZEWALL contains a bioinformatic analysis and gene expression data repertory of cell wall biosynthesis and assembly in maize.
- 735 maize sequences classified into :
     - 174 gene families classified into :
          - 18 functional cell wall-related categories

How was MAIZEWALL built ?

Schematic view

Keyword search
Although many enzymes/genes putatively involved in different aspects of cell wall metabolism have been identified, the precise role of relatively few have been unambiguously demonstrated, and this is especially true in the case in monocots. As a first step to fill this gap, nearly 100 keywords were defined based on current knowledge of genes implicated in cell wall biosynthesis and vascular patterning from all plant species. The keywords fall into different categories including cellulose synthesis, non-cellulosic polysaccharide synthesis, cell wall proteins, lignin/lignans, general phenylpropanoid, transcription factors, etc... Each keyword was used to search NCBI databases to retrieve the corresponding nucleotide and protein sequences and related literature references. Maize genes were retrieved when available. If a maize sequence was not available, we retrieved sequences from other species, especially those for which functional analysis has already proven gene function.
In order to obtain maize homologs, the protein and nucleotide sequences were used to search the most homologous maize sequences using a BLAST program on the Maize GénoplanteInfo Contig database (GPI, Samson et al., 2003). Only contigs with the expected annotations (confirmed by blasting against the SWALL database (Swissprot, Trembl and new)) were integrated into MAIZEWALL.

Homology-based approach from a Zinnia elegans EST subtractive library enriched in genes expressed during secondary wall formation
A Suppressive Substractive Hybridization library (SSH) was constructed from differentiating tracheary elements of Zinnia elegans in vitro (Pesquet et al., 2005). Several time points for library construction were selected : pre-secondary cellulose thickening, pre-lignification and early cell death. The late-stage xylogenesis library (LXL) contained 232 non-redundant Expressed Sequence Tags (ESTs) and each was used to blast Maize GénoplanteInfo Contig database (GPI).

Sample description


Transcriptome analysis has been performed on roots, stem and leaves at the 4-5 leaf stage and internodes at silking. Lignin biochemistry and cytological studies have also been performed on these samples (Guillaumie et al.).

Macroarray construction and hybridization

Schematic view

In order to design a macroarray that guarantees signal specificity and sensitivity, a pilot experiment was carried out on a set of 3 O-methyltransferase genes selected for their known expression profiles. The experiment was designed to optimize the following parameters: 1/ the quantity of cDNA spotted on the membrane, 2/ the source of probe synthesis (total RNA vs polyA RNA), 3/ probe type (reverse-transcribed cDNA vs. random priming), 4/ the length of the Gene Specific Tag (GST) spotted, and 5/ the relative proportion of 3’UTR and coding sequence to insure hybridization specificity within a multigene family. This experiment allowed us to define the following technical parameters for the macroarray as follows : 150-250bp PCR products corresponding to the 3’UTR for each gene was spotted at a concentration of 0.25µg/µl (100nanolitres/spot); the cDNA probe was generated from reverse transcription of total RNA.