Comparative transcriptome profiling of maize coleoptilar nodes during shoot-borne root initiation.

Maize (Zea mays) develops an extensive shoot-borne root system to secure water and nutrient uptake and to provide anchorage in the soil. In this study, early coleoptilar node (first shoot node) development was subjected to a detailed morphological and histological analysis. Subsequently, microarray profiling via hybridization of oligonucleotide microarrays representing transcripts of 31,355 unique maize genes at three early stages of coleoptilar node development was performed. These pairwise comparisons of wild-type versus mutant rootless concerning crown and seminal roots (rtcs) coleoptilar nodes that do not initiate shoot-borne roots revealed 828 unique transcripts that displayed RTCS-dependent expression. A stage-specific functional analysis revealed overrepresentation of "cell wall," "stress," and "development"-related transcripts among the differentially expressed genes. Differential expression of a subset of 15 of 828 genes identified by these microarray experiments was independently confirmed by quantitative real-time-polymerase chain reaction. In silico promoter analyses revealed that 100 differentially expressed genes contained at least one LATERAL ORGAN BOUNDARIES domain (LBD) motif within 1 kb upstream of the ATG start codon. Electrophoretic mobility shift assay experiments demonstrated RTCS binding for four of these promoter sequences, supporting the notion that differentially accumulated genes containing LBD motifs are likely direct downstream targets of RTCS.

The mucilage proteome of maize (Zea mays L.) primary roots.

Maize (Zea mays L.) root cap cells secrete a large variety of compounds including proteins via an amorphous gel structure called mucilage into the rhizosphere. In the present study, mucilage secreted by primary roots of 3-4 day old maize seedlings was collected under axenic conditions, and the constitutively secreted proteome was analyzed. A total of 2848 distinct extracellular proteins were identified by nanoLC-MS/MS. Among those, metabolic proteins (approximately 25%) represented the largest class of annotated proteins. Comprehensive sets of proteins involved in cell wall metabolism, scavenging of reactive oxygen species, stress response, or nutrient acquisition provided detailed insights in functions required at the root-soil interface. For 85-94% of the mucilage proteins previously identified in the relatively small data sets of the dicot species pea, Arabidopsis, and rapeseed, a close homologue was identified in the mucilage proteome of the monocot model plant maize, suggesting a considerable degree of conservation between mono and dicot mucilage proteomes. Homologues of a core set of 12 maize proteins including three superoxide dismutases and four chitinases, which provide protection from fungal infections, were present in all three mucilage proteomes investigated thus far in the dicot species Arabidopsis, rapeseed, and pea and might therefore be of particular importance.

Regulation of the maize (Zea mays L.) embryo proteome by RTCS which controls seminal root initiation.

Seminal roots are initiated at the scutellar node during maize (Zea mays L.) embryo development. The maize mutant rtcs (rootless concerning crown and seminal roots) does not initiate seminal roots while its wild-type siblings form on average 2.9 seminal roots per seedling. In this study, proteome profiles of 25-day-old immature maize embryos were compared between wild-type and rtcs plants via two-dimensional electrophoresis (2-DE). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 23 proteins encoded by 21 different genes that were differentially accumulated between wild-type and rtcs embryos (Fc> or =2; FDR<10%). Among the differentially accumulated proteins, two isoforms of a phosphoglycerate kinase and a malate dehydrogenase were preferentially accumulated in wild-type embryos. Both enzymes are related to the generation of energy-rich ATP or NADPH molecules and are crucial checkpoints of cellular energetics in plants. Comparison of embryonic proteins differentially accumulated between wild-type and rtcs embryos revealed little overlap with proteins differentially accumulated between wild-type and rum1 embryos which also do not initiate seminal roots. This might be due to distinct influences of RTCS and RUM1 on the composition of the embryo proteome, but could also be explained by different stages of embryo development that were analyzed in these studies.

Regulation of the pericycle proteome in maize (Zea mays L.) primary roots by RUM1 which is required for lateral root initiation.

Lateral roots are all roots that are initiated in the pericycle cell layer of other roots during postembryonic development. The maize (Zea mays L.) mutant rum1 (rootless with undetectable meristems 1) does not initiate lateral roots in the primary root. In the present study, two-dimensional electrophoresis proteome profiles of three biological replicates of pericycle cells isolated from the differentiation zone of 2.5-day-old wild-type and rum1 primary roots were generated. This early developmental stage was selected in order to analyze histologically similar cells before the initiation of lateral roots in wild-type primary roots. In total, 418 proteins were reproducibly detected on all six gels after fluorescent staining with Flamingo dye. Among those, twelve proteins were differentially accumulated between wild-type and rum1 pericycle cells (Fc > 2; p < 0.05). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified eight of the twelve proteins. Six proteins were related to metabolism, one protein belonged to the class of disease and defense, and one protein was related to development. Six of the eight proteins have not been previously localized to the pericycle. Moreover, the slight overlap between proteins and transcripts that are differentially accumulated in the maize pericycle between wild-type and rum1 underscores the importance of posttranscriptional protein modifications that cannot be detected on the RNA level. The differential accumulation of proteins in rum1 and wild-type pericycle cells of the primary root suggests that the abundance of these proteins could be regulated by RUM1.

Comparison of maize (Zea mays L.) F1-hybrid and parental inbred line primary root transcriptomes suggests organ-specific patterns of nonadditive gene expression and conserved expression trends.

The phenomenon of heterosis describes the increased agronomic performance of heterozygous F(1) plants compared to their homozygous parental inbred plants. Heterosis is manifested during the early stages of root development in maize. The goal of this study was to identify nonadditive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two-step strategy was used. First, a microarray preselection of nonadditively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high-throughput quantitative real-time (qRT)-PCR experiments. Initial microarray experiments identified 1941 distinct microarray features that displayed nonadditive gene expression in at least 1 of the 12 analyzed hybrids compared to the midparent value of their parental inbred lines. Most nonadditively expressed genes were expressed between the parental values (>89%). Comparison of these 1941 genes with nonadditively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance. This finding suggests organ-specific patterns of nonadditively expressed genes. qRT-PCR analyses of 64 of the 1941 genes in four different hybrids revealed conserved patterns of nonadditively expressed genes in different hybrids. Subsequently, 22 of the 64 genes that displayed nonadditive expression in all four hybrids were analyzed in 12 hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all 12 hybrids and might thus play a protective role in heterosis-related antioxidative defense in the primary root of maize hybrids. The findings of this study are consistent with the hypothesis that both global expression trends and the consistent differential expression of specific genes contribute to the organ-specific manifestation of heterosis.

Proteomic dissection of plant development.

Plant development is controlled by complex endogenous genetic programs and responses to environmental cues. Proteome analyses have recently been introduced to plant biology to identify proteins instrumental in these developmental processes. To date most plant proteome studies have been employed to generate reference maps of the most abundant soluble proteins of plant organs at a defined developmental stage. However, proteomics is now also utilized for genetic studies comparing the proteomes of different plant genotypes, for physiological studies analyzing the influences of exogenous signals on a particular plant organ, and developmental studies investigating proteome changes during development. Technical advances are now beginning to allow a proteomic dissection of individual cell types, thus greatly increasing the information revealed by proteome analyses.