Salinity induces specific metabolic changes in sugarcane shoot explants in temporary immersion bioreactors

There is a great demand of salt-tolerant sugarcane planting material in Cuba. Temporary immersion bioreactors (TIB) are effective to significantly increase sugarcane in vitro shoot proliferation rate from 1:4 in conventional containers to about 1:35. Sugarcane micropropagation in TIBs under NaCl stress may help screen mutants with salinity tolerance. We developed the experiment shown here to identify a NaCl concentration able to stress shoot in TIBs. At 30 days of culture initiation with different NaCl levels (0 - 200 mM), explant multiplication rate, shoot cluster fresh mass, and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. Content of soluble phenolics in the culture medium was also evaluated. Addition of NaCl decreased shoot multiplication rate and fresh mass. Other statistically significant differences were recorded but the most important were noted in the increased contents of carotenoids, malondialdehyde, other aldehydes and soluble phenolics in the plants, and in the soluble phenolics in the culture medium. This research may be useful for future experiments of in vitro selection of new sugarcane genetic materials with NaCl tolerance. Fifty percent of multiplication rate was reduced with 89 mM NaCl which can be used to stress shoots during micropropagation in TIBs and eventually detect mutants with salt tolerance

Cell Cycle Modulation in the Response of the Primary Root of Arabidopsis to Salt Stress

Salt stress inhibits plant growth and development. We investigated the importance of cell cycle regulation in mediating the primary root growth response of Arabidopsis to salt stress. When seedlings were transferred to media with increasing concentrations of NaCl, root growth rate was progressively reduced. At day 3 after transfer of seedlings to growth medium containing 0.5% NaCl the primary roots grew at a constant rate well below that prior to the transfer, whereas those transferred to control medium kept accelerating. Kinematic analysis revealed that the growth reduction of the stressed roots was due to a decrease in cell production and a smaller mature cell length. Surprisingly, average cell cycle duration was not affected. Hence, the reduced cell production was due to a smaller number of dividing cells, i.e. a meristem size reduction. To analyze the mechanism of meristem size adaptation prior to day 3, we investigated the short-term cell cycle events following transfer to saline medium. Directly after transfer cyclin-dependent kinase (CDK) activity and CYCB1;2 promoter activity were transiently reduced. Because protein levels of both CDKA;1 and CDKB1;1 were not affected, the temporary inhibition of mitotic activity that allows adaptation to the stress condition is most likely mediated by posttranslational control of CDK activity. Thus, the adaptation to salt stress involves two phases: first, a rapid transient inhibition of the cell cycle that results in fewer cells remaining in the meristem. When the meristem reaches the appropriate size for the given conditions, cell cycle duration returns to its default

STUNTED PLANT 1 Mediates Effects of Cytokinin, But Not of Auxin, on Cell Division and Expansion in the Root of Arabidopsis

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 μm zeatin) or auxin (30 nm 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm(−1) h(−1)) and cell division (cells cell(−1) h(−1)). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement

Can Meristematic Activity Determine Variation in Leaf Size and Elongation Rate among Four Poa Species? A Kinematic Study

We studied inherent variation in final leaf size among four Poa spp. that live at different elevations. The average final length of leaf 7 of the main stem of the smallest species (Poa alpina) was only one-half that of the largest species (Poa trivialis); it was correlated with leaf elongation rate, but not with the duration of leaf elongation. A faster rate of leaf elongation rate was associated with (a) larger size of the zone of cell expansion, and (b) faster rates of cell production (per cell file) in the meristem, which in turn were due to greater numbers of dividing cells, whereas average cell division rates were very similar for all species (except Poa annua). Also we found that the proliferative fraction equaled 1 throughout the meristem in all species. It was remarkable that rates of cell expansion tended to be somewhat higher in the species with slower growing leaves. We discuss the results by comparing the spatial and material viewpoints, which lead to different interpretations of the role of cell division. Although the presented data do not strictly prove it, they strongly suggest a regulatory role for cell division in determining differences in growth rate among the present four Poa spp

Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity of Cellulose Microfibril Alignment among Cells in the Root of Arabidopsis

To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule inhibitor, oryzalin. After 2 d of treatment, roots grow at approximately steady state. At that time, the spatial profiles of relative expansion rate in length and diameter were quantified, and roots were cryofixed, freeze-substituted, embedded in plastic, and sectioned. The angular distribution of microtubules as a function of distance from the tip was quantified from antitubulin immunofluorescence images. In alternate sections, the overall amount of alignment among microfibrils and their mean orientation as a function of position was quantified with polarized-light microscopy. The spatial profiles of relative expansion show that the drug affects relative elongation and tangential expansion rates independently. The microtubule distributions averaged to transverse in the growth zone for all treatments, but on oryzalin the distributions became broad, indicating poorly organized arrays. At a subcellular scale, cellulose microfibrils in oryzalin-treated roots were as well aligned as in controls; however, the mean alignment direction, while consistently transverse in the controls, was increasingly variable with oryzalin concentration, meaning that microfibril orientation in one location tended to differ from that of a neighboring location. This conclusion was confirmed by direct observations of microfibrils with field-emission scanning electron microscopy. Taken together, these results suggest that cortical microtubules ensure microfibrils are aligned consistently across the organ, thereby endowing the organ with a uniform mechanical structure