Mohammed Bajji

The use of the electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in durum wheat

This work was carried out to adapt the electrolyte leakage technique todurum wheat and then to evaluate its relevance in the assessment of the cellmembrane stability as a mechanism of water stress tolerance in this species.Themethod currently used is based on in vitro desiccation ofleaf tissues by a solution of polyethylene glycol (PEG) and a subsequentmeasurement of electrolyte leakage into deionised water. It consists of threesuccessive steps: (1) a washing treatment to remove solutes from both leafsurfaces and cells damaged by cutting; (2) a stress period during which theleaftissues are plunged in a PEG-solution and (3) a rehydration period during whichafter-effects of the stress are evaluated. During the washing period, the majorpart of electrolytes was removed within 15 min. Varying the stressconditions influenced both the percent and the kinetics of electrolyte leakageduring rehydration. Electrolyte leakage exhibited a characteristic patternreflecting the condition of cellular membranes (repair and hardening). Inpractice, we recommend a 15-minute washing time, a10-hour stress period and 4 h of rehydration. Theextent of the cell membrane damage not only correlated well with the growthresponses of wheat seedlings belonging to various cultivars to withholdingwaterbut also with the recognised field performances of these cultivars. Therelativeproportion of endogenous ions lost in the effusate during the rehydration stepmay vary strongly according to the element analysed and the precise nutritionalstatus of the plant should therefore be considered. However, an increase ininorganic ion leakage does not fully explain the recorded PEG-induced increasein electrical conductivity (EC) during the subsequent rehydration step andorganic ions are probably also involved in such an increase.

Water deficit effects on solute contribution to osmotic adjustment as a function of leaf ageing in three durum wheat (Triticum durum Desf.) cultivars performing differently in arid conditions.

A greenhouse study was carried out using three durum wheat (Triticum durum Desf.) cultivars differing in their field performances under arid conditions (Kabir 1, poor yield stability; Omrabi 5, high yield stability and Haurani, landrace well adapted to drought). Water stress was imposed by withholding water at the seedling stage. Water potential (Psi(w)), relative water content (RWC), stomatal resistance (SR), and changes in solute concentrations were quantified: (1) as a function of leaf development during the stress period; and (2) in young expanded and growing leaves harvested at the end of the stress treatment. Psi(w), RWC and SR were almost unaffected by leaf age in controls. In contrast, solute concentrations appeared to vary in the course of leaf development. During the stress treatment, Psi(w) and RWC decreased and SR increased in all cultivars; the changes were most often largest in Omrabi 5, lowest in Haurani and intermediate in Kabir 1. Water stress also increased sugar and proline concentrations and decreased nitrate levels. Young expanded and growing leaves differed in terms of Psi(w), RWC and osmotic adjustment (OA). The capacity of OA was greater in growing than in expanded leaves, especially in the two cultivars best adapted to aridity, and allowed turgor maintenance in these genotypes. Sugars were the main solutes that contributed to OA particularly in growing leaves followed by proline and then quaternary ammonium compounds. The contributions of these organic solutes to OA tended to be higher in Omrabi 5 and in Haurani than in Kabir 1. Inorganic solutes, however, did not seem to play an important role in OA despite their high proportion in total solutes.

Salt stress effects on roots and leaves of Atriplex halimus L. and their corresponding callus cultures

Salt stress effects on growth, osmotic adjustment, mineral and organic contents and soluble peroxidase activities were determined in roots and leaves of Atriplex halimus and their corresponding callus cultures. Low NaCl doses (150 mM) promoted shoot growth, corroborating the halophilic nature of this species; in these stress conditions, Na+ concentration markedly increased in the leaves indicating that salinity resistance was not associated with the ability of the plants to restrict sodium accumulation in the aerial part. Whole organs and their corresponding calli were able to cope with high NaCl doses but there was no clear correspondence between the physiological behaviour of cell culture and whole plant. For several physiological parameters (osmotic potential (Psi s), mineral content, proline accumulation), roots were less affected by NaCl than leaves while both root and leaf calli behaved in the same way in response to salinity. NaCl-induced modifications of the recorded parameters are discussed in relation to the mechanisms of salinity resistance in this species. Evidence indicated the existence of a cellular basis for salinity resistance in A. halimus, but the expression of this cellular property at organ level appeared to be masked by the physiological complexity of the intact plant and the nature of the whole organ response was apparently determined primarily by regulation mechanisms assigned by the differentiated tissue organisation

Effect of water stress on growth, Na+ and K+ accumulation and water use efficiency in relation to osmotic adjustment in two populations of Atriplex halimus L.

The effect of water stress on growth, Na+ and K+ accumulation and water utilization was investigated in plants of two populations of Atriplex halimus L. originating from Kairouan (Tunisia) and Tensift (Morocco). Water deficit was applied by withholding water for 22 days. All plants remained alive until the end of the treatment although growth was strongly reduced in both populations. Water stress decreased CO2 assimilation in saturating conditions, mainly in the population obtained from Kairouan, suggesting an impact of drought on the dark phase of photosynthesis, beside a decrease in stomatal conductance which was recorded mainly in the population obtained from Tensift. The two studied populations did not differ in their water consumption, as indicated by similar soil gravimetric water content and plant transpiration. However, water use efficiency increased under stress conditions in the population from Tensift but not in the population from Kairouan. Thelatter population displayed a larger capacity for osmotic adjustment. A drought-induced specific increase in Na+ concentration was also reported in both populations. It is concluded that in A. halimus, water stress resistance estimated in terms of biomass production, could be associated with higher WUE rather than with with a greater osmotic adjustment and that sodium may assume a specific physiological function in this xerohalophytic C4 species.

Physiological changes after exposure to and recovery from polyethylene glycol-induced water deficit in callus cultures issued from durum wheat (Triticum durum Desf.) cultivars differing in drought resistance

Calli obtained from three durum wheat (Triticum durum Desf.) cultivars with different drought resistance levels were exposed for 30 days to a polyethylene glycol-induced water deficit followed by 30 days of recovery. Relative growth rate, percent dry weight, osmotic potential and the changes in inorganic and organic solutes were determined at the end of both the stress and the recovery periods. After the stress period, calli derived from the drought resistant cultivars, particularly Omrabi 5 and to some extent Haurani, showed a less reduced relative growth rate, lower osmotic potential and higher proline and other amino acid accumulations as compared with the sensitive cultivar Kabir I. Drought resistance could also be associated with a better protection of enzymes involved in nitrogen metabolism. At the end of the recovery period, most of the quantified parameters, except osmotic potential, recovered completely, indicating the reversibility of the changes induced by water deficit at the plant cell level in durum wheat. Our data show that a correlation exists between performances of the cultivars under drought and the responses of callus cultures to PEG-induced water deficit; this suggests that: in durum wheat the degree of resistance to drought at the plant level depends, at least in part, on the existence of mechanisms operating at the cellular level.

Physiological changes after exposure to and recovery from polyethylene glycol-induced water deficit in roots and leaves of durum wheat (Triticum durum Desf.) cultivars differing in drought resistance

The effects of water stress on the behaviour of hydroponically-grown seedlings of 3 durum wheat ( Triticum durum Desf.) cvs. differing in drought resistance (Kabir 1, drought sensitive; Omrabi 5 and Haurani, drought resistant) were investigated under controlled environmental conditions. Fresh weight (FW), water content (WC), osmotic potential (Psi(s)), organic and inorganic solute concentrations in roots and shoots were measured after 7 days of exposure to polyethylene glycol (PEG at 0, 10 or 20 %) and after 2 and 7 days of recovery following stress relief. Water stress decreased FW WC and Psi s, and increased root to shoot ratio (R/S) and soluble sugar, proline and other amino acid concentrations in both roots and leaves of all cvs. Water stress effect on inorganic solutes was different depending on the ion nature and on the plant part. Most of the significant changes were recorded in response to 20 % PEG. Roots and shoots roughly reacted in the same way to water stress but differed in terms of the intensity and rapidity of the responses. After stress relief, almost all parameters returned to control level; however, for some of them, roots and shoots differed in the rapidity of their recovery. Growth parameters as well as organic solute accumulation revealed differences in the behaviour of the 3 different cvs. when confronted to water stress. Our results demonstrated the high plasticity of durum wheat at the vegetative stage and suggest that the maintenance of higher root growth and osmotic adjustment in water stress conditions could confer an improved resistance of this species to drought.

Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4 generations. Differences in electrolyte leakage, chlorophyll fluorescence (F-v/F-m), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4 parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells

Aquaporins (AQPs) are water channels that allow cells to rapidly alter their membrane water permeability. A convenient model for studying AQP expression and activity regulation is Black Mexican Sweet (BMS) maize cultured cells. In an attempt to correlate membrane osmotic water permeability coefficient (P(f)) with AQP gene expression, we first examined the expression pattern of 33 AQP genes using macro-array hybridization. We detected the expression of 18 different isoforms representing the four AQP subfamilies, i.e. eight plasma membrane (PIP), five tonoplast (TIP), three small basic (SIP) and two NOD26-like (NIP) AQPs. While the expression of most of these genes was constant throughout all growth phases, mRNA levels of ZmPIP1;3, ZmPIP2;1, ZmPIP2;2, ZmPIP2;4 and ZmPIP2;6 increased significantly during the logarithmic growth phase and the beginning of the stationary phase. The use of specific anti-ZmPIP antisera showed that the protein expression pattern correlated well with mRNA levels. Cell pressure probe and protoplast swelling measurements were then performed to determine the P(f). Interestingly, we found that the P(f) were significantly increased at the end of the logarithmic growth phase and during the steady-state phase compared to the lag phase, demonstrating a positive correlation between AQP abundance in the plasma membrane and the cell P(f).