Analysis of Microtubule-Associated-Proteins during IBA-Mediated Adventitious Root Induction Reveals KATANIN Dependent and Independent Alterations of Expression Patterns.

Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs.

Dissecting the contribution of microtubule behaviour in adventitious root induction.

Induction of adventitious roots (ARs) in recalcitrant plants often culminates in cell division and callus formation rather than root differentiation. Evidence is provided here to suggest that microtubules (MTs) play a role in the shift from cell division to cell differentiation during AR induction. First, it was found that fewer ARs form in the temperature-sensitive mutant mor1-1, in which the MT-associated protein MOR1 is mutated, and in bot1-1, in which the MT-severing protein katanin is mutated. In the two latter mutants, MT dynamics and form are perturbed. By contrast, the number of ARs increased in RIC1-OX3 plants, in which MT bundling is enhanced and katanin is activated. In addition, any1 plants in which cell walls are perturbed made more ARs than wild-type plants. MT perturbations during AR induction in mor1-1 or in wild-type hypocotyls treated with oryzalin led to the formation of amorphous clusters of cells reminiscent of callus. In these cells a specific pattern of polarized light retardation by the cell walls was lost. PIN1 polarization and auxin maxima were hampered and differentiation of the epidermis was inhibited. It is concluded that a fine-tuned crosstalk between MTs, cell walls, and auxin transport is required for proper AR induction.

Gene expression profiling in juvenile and mature cuttings of Eucalyptus grandis reveals the importance of microtubule remodeling during adventitious root formation.

BACKGROUND: The ability to form adventitious roots (AR) is an economically important trait that is lost during the juvenile-to-mature phase change in woody plants. Auxin treatment, which generally promotes rooting in juvenile cuttings, is often ineffective when applied to mature cuttings. The molecular basis for this phenomenon in Eucalyptus grandis was addressed here. RESULTS: A comprehensive microarray analysis was performed in order to compare gene-expression profiles in juvenile and mature cuttings of E. grandis, with or without auxin treatment on days, 0, 1, 3, 6, 9 and 12 post AR induction. Under these conditions AR primordia were formed only in auxin-treated juvenile cuttings. However, clustering the expression profiles revealed that the time after induction contributed more significantly to the differences in expression than the developmental phase of the cuttings or auxin treatment. Most detected differences which were related to the developmental phase and auxin treatment occurred on day 6, which correlated with the kinetics of AR-primordia formation. Among the functional groups of transcripts that differed between juvenile and mature cuttings was that of microtubules (MT). The expression of 42 transcripts annotated as coding for tubulin, MT-associated proteins and kinesin motor proteins was validated in the same RNA samples. The results suggest a coordinated developmental and auxin dependent regulation of several MT-related transcripts in these cuttings. To determine the relevance of MT remodeling to AR formation, MTs were subjected to subtle perturbations by trifluralin, a MT disrupting drug, applied during auxin induction. Juvenile cuttings were not affected by the treatment, but rooting of mature cuttings increased from 10 to more than 40 percent. CONCLUSIONS: The data suggest that juvenile-specific MT remodeling is involved in AR formation in E. grandis.

Profiling microRNAs in Eucalyptus grandis reveals no mutual relationship between alterations in miR156 and miR172 expression and adventitious root induction during development.

BACKGROUND: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. RESULTS: We characterized the population of miRNAs of Eucalyptus grandis and compared the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. CONCLUSIONS: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability.

Microarray analysis revealed upregulation of nitrate reductase in juvenile cuttings of Eucalyptus grandis, which correlated with increased nitric oxide production and adventitious root formation.

The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), which is involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO was observed upon excision, which was higher in juvenile cuttings than in mature ones. Treatment with an NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production in intact plants. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to increased ability to produce NO and to form adventitious roots. Arabidopsis transgenic plants constantly expressing EgNIA did not exhibit a significantly higher lateral or adventitious root formation, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root differentiation.