Maria-Jesus Cornejo

Common and distinctive responses of rice seedlings to saline- and osmotically-generated stress

Drought and salinity are the major abiotic factors limiting productivityin rice (Oryza sativa L.). Although both generate osmoticstress, ion toxicity is an additional and important component of salinity. Tostudy the morphological and anatomical responses to those types of stress, weused in vitro grown rice seedlings. Based on an initialscreening of several non-penetrating osmotica on seedling growth, we selectedsorbitol to compare its osmotic effects during seedling development with thosegenerated by NaCl stress. At comparable levels of osmolality, the reduction inroot and leaf growth as well as their delayed development were similar for bothsaline- and osmotically-generated stress. Some changes observed in root anatomyand most of the variations in leaf anatomy features caused by the treatmentscould be ascribed to osmotic stress. However, there were evident differences inthe morphology of the root system as well as in chlorophyll levels as afunctionof the stress treatment. Furthermore, the larger size of epidermal andbulliformcells was distinctively related to saline stress. The results obtained providetools for the in vitro identification of either specificorcross-tolerant phenotypes.

Differential salinity-induced variations in the activity of H⁺-pumps and Na⁺/H⁺ antiporters that are involved in cytoplasm ion homeostasis as a function of genotype and tolerance level in rice cell lines.

The characterisation of cellular responses to salinity in staple crops is necessary for the reliable identification of physiological markers of salinity tolerance. Under saline conditions, variations in proton gradients that are generated by membrane-bound H⁺ pumps are crucial for maintaining cytoplasm homeostasis. We examined short (15 h) and longer term effects (4 days) of NaCl stress on the H⁺ pumping activities that are associated with the plasma membrane (P-ATPase) and the tonoplast (V-ATPase and V-PPase) in rice (Oryza sativa L.) callus lines that displayed different levels of NaCl tolerance and were established from two japonica rice cultivars. The applied stress conditions were based on those that were used in the induction of a stress-responsive polyubiquitin gene promoter (UBI1) in transgenic rice calli. The most remarkable effect of NaCl stress on H⁺ pumping was the rapid activation of tonoplast-bound pumps; this was particularly observed in cv. Bomba, in which the response of the P-ATPase was slower and showed a higher level of activity after 4 days of stress. The responses were cultivar-dependent; however, in general, a stronger activation occurred in the lines that had a higher tolerance (L-T) than in the less-tolerant (L-S) lines. Substrate hydrolysis was less affected than H⁺ pumping, and it yielded higher H⁺/substrate coupling ratios, which is indicative of an enhanced H⁺ pumping efficiency under saline conditions. The Na⁺/H⁺ antiport activity was generally limited to salt-stressed calli, and higher values and stronger activation of the tonoplast antiporter were observed in the L-T lines than in the L-S lines. The results that were obtained with the NaCl-stressed transgenic lines confirmed the close relationship between metabolic activity, H⁺ pumping and the induction of Na⁺/H⁺ exchange activities.

Uptake of endocytic markers by rice cells: variations related to the growth phase.

Endocytosis is now considered a basic cellular process common to plant cells. Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear. We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers. First, we show that markers are internalized by rice cells and appear in intracellular membranes. The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin. Thus, it shows the properties of a receptor-mediated process. We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically. Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake. Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake. On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake. The possible causes for this arrest of endocytosis are discussed.

Gibberellic acid stimulates lipid metabolism in barley aleurone protoplasts

Abstract Changes in the lipid composition of barley aleurone protoplasts following incubation in the presence or absence of gibberellic acid (GA) were studied and correlated with cell development. Analysis of neutral lipids in protoplast extracts and purified lipid bodies provide evidence for the strong effect of GA on the mobilization of storage lipids. In vivo protoplast staining with the membrane marker N -phenyl-l-naphthylamine (NPN) and phospholipid determinations of protoplast extracts show an increase in membrane lipids during protoplast incubation. GA stimulates phospholipid metabolism by increasing the synthesis of phosphatidyl ethanolamine and phosphatidyl choline, the major phospholipid constituents of the endoplasmic reticulum.

Phenotypic indicators of NaCl tolerance levels in rice seedlings: variations in development and leaf anatomy

The aim of this research is the early identification of distinctive responses to NaCl in rice cultivars that would indicate further stress-related effects in mature plants. For this purpose, we analysed some developmental and anatomical features in control and NaCl-stressed seedlings of two Japonica rice cultivars (Bomba and Bahia). Responses ascribed to osmotic stress were differentiated from those related to the ionic component of salinity by using in parallel a non-penetrating osmoticum (sorbitol). The general patterns of reduction in growth and variations in anatomical features of second leaf sections were similar in both cultivars. The main difference between them was the intensity of the response as a function of the stress agent. In general, the effect of NaCl was significantly stronger than that of sorbitol in cv. Bomba, whereas in cv. Bahia the effects of both stress agents were comparable. In this regard, the size of epidermal and bulliform cells as well as dimensions related to the vascular system, including xylem vessels, increased significantly in NaCl-stressed cv. Bomba leaves. This enlargement of xylem vessels agrees with the observed decrease in the rate of eosin transport and appears to be a distinctive anatomical indicator of NaCl sensitivity. The further impact of salinity on grain yield was proved to be stronger in cv. Bomba plants than in those of cv. Bahia.

Rice protoplasts isolated directly from mature-embryo-derived calli

Mature-embryo-derived calli of japonica rice (Oryza sativa L) Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisfactory yield of 5.20×106–8.96×106 protoplasts/g FW (fresh weight). The remaining three callus lines initiated from seeds of cryopreserved-callus-derived plants had rooty calli, resulting in low yield of protoplasts and a large number of isolated banana-shape intact cells. Viability of protoplasts ranged 87.46%–94.15%. The average size of protoplasts was 207.49–379.04 μm2 in different callus lines. Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts.

The Effect of Secretion on Lipids and their Distribution in Barley Aleurone Protoplasts

Barley aleurone protoplasts, when exposed to Ga3 and Ca2+ synthesize and secrete the hydrolytic enzyme α -amylase (Bush et al., 1986). At the same time, protoplasts undergo important structural changes which reflect the elaboration of the membrane system involved in protein synthesis and secretion. The mobilization of lipid reserves may be a preliminar step which would provide energy as well as several components for the synthesis of membrane lipids.