Molecular characterizaton of root hair elongation in maize
Final Report Abstract
Root hairs are unicellular, tubular extensions of epidermal cells securing optimal water and nutrient supply of plants by increasing the absorbing surface of roots. Therefore, root hairs of crop plants are of eminent agronomic importance. In this project the genes roothairless5 and roothairless6, both involved in root hair elongation were cloned and functionally characterized. Moreover, reference proteome and transcriptome maps of maize root hairs were generated. Root hairs of the rth5 mutant are significantly impaired in elongation, displaying an average length of only 4% compared to wild-type root hairs. Similarly, root hairs of the mutant rth6 are also severely impeded in elongation and grow to only about 5% of the wild-type root hair length. Both mutations affect root hairs on all major root types including primary, seminal and shoot-borne roots. Molecular cloning demonstrated that rth5 encodes a transmembrane NADPH oxidase. Cloning of the rth6 revealed that this gene encodes the CELLULOSE SYNTHASE-LIKE D (CslD) protein CslD5. Both rth5 and rth6 are preferentially expressed in root hairs and map to monocot specific subclades of their phylogenetic trees. The genes rth5, rth6 and the previously cloned gene rth3 are functionally linked. The NADPH oxidase RTH5 produces apoplastic superoxide, which results in cell-wall loosening via hydroxyl radicals and also controls the expression of cellulose biosynthesis genes. After cell wall loosening, the transmembrane protein RTH6 is arranged in rosettes and synthesizes cellulose at the plasma membrane which is extruded to the inner side of the cell wall in the root hair tip and thus reinforces the tubular shaft, while the GPI-anchored COBRA-like cell wall protein RTH3 is involved in the organization of the synthesized cellulose. To dissect root hair formation and study factors involved in their functionality on the protein level, the most abundant soluble proteins were isolated from wild-type root hairs digested by trypsin and identified using NanoLC-MS/MS (nano liquid chromatography- tandem mass spectrometry) and assigned to unique proteins encoded by the maize genome. A total of 2,573 soluble root hair proteins represented by at least two peptides were identified. This study defined the proteomic landscape of maize root hairs and their functionality. The most abundant functional class was related to protein biosynthesis, degradation, and localization and comprised 25% of all identified proteins. Moreover, known homologs of root proteins involved in root hair formation in Arabidopsis were identified. A comparison with 1492 proteins of the soybean reference genome revealed conserved but also unique functions of root hairs in monocot maize versus dicot soybean plants. To generate a reference transcriptome map and to identify root hair specific genes, the transcriptomes of 3-day-old root hairs and primary roots without root hairs were compared. Among 27,155 expressed genes 3% were exclusively active in root hairs. Although most genes were expressed in primary roots without root hairs and in root hairs most genes expressed in the dataset (20,470 genes) were differentially expressed between both tissues. More genes were preferentially expressed in roots without root hairs (14,881 genes) than in root hairs (3436 genes), indicating a lower transcriptomic diversity in root hairs than in roots without root hairs. For Arabidopsis genes known to be involved in root hairs homologs were identified in maize and their expression levels were compared. The maize root hair transcriptome revealed similarities, but also differences to the Arabidopsis root hair transcriptome. While GO annotation of the maize root hair transcriptome for instance highlights the importance of energy metabolism in root hairs, this aspect was much less pronounced in the Arabidopsis root hair transcriptome. In summary, this project cloned and characterized two major check points of maize root hair elongation rth5 and rth6. Moreover, the generation of root hair proteome and transcriptome maps provided a starting point for future studies of candidate genes involved in maize root hair formation.
Publications
- (2009) Genetic and genomic dissection of maize root development and architecture. Curr. Opin. Plant Biol. 12: 172-177
Hochholdinger F., Tuberosa R.
- (2009) Molecular and genetic dissection of cereal root development. Ann. Plant Rev. 37: 175–191
Hochholdinger F., Zimmermann R.
- (2009) The Maize Root System: Morphology, Anatomy and Genetics. In: The Handbook of Maize. Springer, New York, 145-160
Hochholdinger F.
- (2011) Conserved and unique features of the maize root hair proteome. J. Proteome Res. 10: 2525-2537
Nestler J., Schütz W., Hochholdinger F.
- (2013) Genetic analysis of maize root development. In: Plant roots the hidden half. 4th ed., Marcel Dekker, New York, 10: 1-8
Hochholdinger F., Feix G.
- (2013) Genetic control of root organogenesis in cereals. Meth. Mol. Biol. 959: 69-81
Marcon C., Paschold A., Hochholdinger F.
- (2013) Plant Root Development, Genetics and Genomics Of. In: Brenner's Encyclopedia of Genetics, Elsevier, New York, 5: 349-352
Hochholdinger F., Nestler J.
- (2014) Association analysis of single nucleotide polymorphisms in candidate genes with root traits in maize (Zea mays L.) seedlings. Plant Sci. 224: 9-19
Kumar B., Abdel-Ghani A.H., Pace J., Reyes-Matamoros J., Hochholdinger F., Lübberstedt T.
(See online at https://doi.org/10.1016/j.plantsci.2014.03.019) - (2014) Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase. Plant J. 79: 729-740
Nestler J., Liu S., Wen T.-J., Paschold A., Marcon C., Tang H.M., Li D., Li L., Meeley R., Sakai H., Bruce W., Schnable P.S., Hochholdinger F.
(See online at https://doi.org/10.1111/tpj.12578) - (2016) Characterization of maize roothairless6 which encodes a D- type cellulose synthase and controls the switch from bulge formation to tip growth. Sci. Rep. 6: 34395.
Li L., Hey S., Liu S., Liu Q., McNinch C., Hu H.C., Wen T.J., Marcon C., Paschold A., Bruce W., Schnable P.S., Hochholdinger F.
(See online at https://doi.org/10.1038/srep34395) - (2016) Untapping root system architecture for crop improvement. J. Exp. Bot. 67: 4431-4433
Hochholdinger F.
(See online at https://doi.org/10.1093/jxb/erw262)
