Stanley Lutts

An improved protocol to study the plant cell wall proteome

Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.

Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.)

In order to determine the relative importance of ionic toxicity versus the osmotic component of salt stress on germination in durum wheat (Triticum durum Desf.), seeds of three cultivars differing in their salt and drought resistance (Omrabi-5, drought-resistant; Belikh, salt-resistant and Cando, salt-sensitive) were incubated in various iso-osmotic solutions of NaCl, mannitol and polyethylene-glycol (PEG) (osmotic potential of −0.15 (control solution) −0.58, −1.05 or −1.57 MPa). Moderate stress intensities only delayed germination, whereas the highest concentration of NaCl and PEG reduced final germination percentages. PEG was the most detrimental solute, while mannitol had no effect on final germination percentages. All osmotica reduced endosperm starch and soluble sugars content as well as α-amylase activities recorded after 48 h of treatment while β-amylase activities were, in contrast, slightly stimulated in all cultivars. Deleterious effects of NaCl and PEG were higher on isolated embryos germinated onto an in vitro Linsmaier and Skoog (LS) medium comparatively to whole seeds. All PEG-treated embryos, however, recovered after the stress relief while NaCl-treated embryos exhibited a lower rate of recovery and some extent of abnormal germination after rinsing. It was concluded that stress inhibition of germination could not be attributed to an inhibition of mobilisation of reserves and that the main effect of PEG occurred via an inhibition of water uptake while detrimental effects of NaCl may be linked to long-term effects of accumulated toxic ions. The behaviour of the three cultivars during germination did not fully reflect their mean level of putative stress resistance in field conditions and germination is, therefore, not recommended as a reliable selection criterion for breeding purposes.

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.

Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L) cultivars differing in salinity resistance

Salt-resistant rice cultivars Nona Bokra and IR 4630 exposed at the seedling stage during one or two weeks to 0, 20, 30, 40 or 50 mM NaCl accumulated less Na, Cl, Zn and proline and more K at root and shoot levels than salt-sensitive I Kong Pao and IR 31785. Aiwu, a moderately resistant genotype, exhibited an intermediate behaviour. P transport from root to shoot was inhibited in the most sensitive cultivar IR 31785. Accumulation of Na and Cl and decrease in K content at the shoot level were restricted to the oldest leaves in salt-resistant genotypes while proline accumulated in the youngest leaves in all cultivars. In the presence of NaCl, the osmotic potentials of the roots and of the oldest and youngest leaves were lower in the salt-resistant than in the salt-sensitive genotypes, differences among genotypes increasing with stress intensity. Proline did not appear to be involved in osmotic adjustment in salt-stressed rice plants and the significance of its accumulation is discussed in relation to salinity resistance.

Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants

Following exposure to salinity, the root/shoot ratio is increased (an important adaptive response) due to the rapid inhibition of shoot growth (which limits plant productivity) while root growth is maintained. Both processes may be regulated by changes in plant hormone concentrations. Tomato plants (Solanum lycopersicum L. cv Moneymaker) were cultivated hydroponically for 3 weeks under high salinity (100 mM NaCl) and five major plant hormones (abscisic acid, ABA; the cytokinins zeatin, Z, and zeatin-riboside, ZR; the auxin indole-3-acetic acid, IAA; and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, ACC) were determined weekly in roots, xylem sap, and leaves. Salinity reduced shoot biomass by 50–60% and photosynthetic area by 20–25% both by decreasing leaf expansion and delaying leaf appearance, while root growth was less affected, thus increasing the root/shoot ratio. ABA and ACC concentrations strongly increased in roots, xylem sap, and leaves after 1 d (ABA) and 15 d (ACC) of salinization. By contrast, cytokinins and IAA were differentially affected in roots and shoots. Salinity dramatically decreased the Z+ZR content of the plant, and induced the conversion of ZR into Z, especially in the roots, which accounted for the relative increase of cytokinins in the roots compared to the leaf. IAA concentration was also strongly decreased in the leaves while it accumulated in the roots. Decreased cytokinin content and its transport from the root to the shoot were probably induced by the basipetal transport of auxin from the shoot to the root. The auxin/cytokinin ratio in the leaves and roots may explain both the salinity-induced decrease in shoot vigour (leaf growth and leaf number) and the shift in biomass allocation to the roots, in agreement with changes in the activity of the sink-related enzyme cell wall invertase.

Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile

Growth, in particular reorganization of the root system architecture, mineral homeostasis and root hormone distribution were studied in Arabidopsis thaliana upon copper excess. Five-week-old Arabidopsis plants growing in hydroponics were exposed to different Cu(2+) concentrations (up to 5 muM). Root biomass was more severely inhibited than shoot biomass and Cu was mainly retained in roots. Cu(2+) excess also induced important changes in the ionome. In roots, Mg, Ca, Fe and Zn concentrations increased, whereas K and S decreased. Shoot K, Ca, P, and Mn concentrations decreased upon Cu(2+) exposure. Further, experiments with seedlings vertically grown on agar were carried out to investigate the root architecture changes. Increasing Cu(2+) concentrations (up to 50 muM) reduced the primary root growth and increased the density of short lateral roots. Experiment of split-root system emphasized a local toxicity of Cu(2+) on the root system. Observations of GUS reporter lines suggested changes in auxin and cytokinin accumulations and in mitotic activity within the primary and secondary root tips treated with Cu(2+). At toxic Cu(2+) concentrations (50 muM), these responses were accompanied by higher root apical meristem death. Contrary to previous reports, growth on high Cu(2+) did not induce an ethylene production. Finally lignin deposition was detected in Cu(2+)-treated roots, probably impacting on the translocation of nutrients. The effects on mineral profile, hormonal status, mitotic activity, cell viability and lignin deposition changes on the Cu(2+)-induced reorganization of the root system architecture are discussed.

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.

Hormonal changes during salinity-induced leaf senescence in tomato (Solanum lycopersicum L.)

Leaf senescence is one of the most limiting factors to plant productivity under salinity. Both the accumulation of specific toxic ions (e.g. Na+) and changes in leaf hormone relations are involved in the regulation of this process. Tomato plants (Solanum lycopersicum L. cv Moneymaker) were cultivated for 3 weeks under high salinity (100 mM NaCl) and leaf senescence-related parameters were studied during leaf development in relation to Na+ and K+ contents and changes in abscisic acid (ABA), cytokinins, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the auxin indole-3-acetic acid (IAA). Na+ accumulated to a similar extent in both leaves 4 and 5 (numbering from the base of the plant) and more quickly during the third week, while concurrently K+ contents sharply decreased. However, photosystem II efficiency, measured as the Fv/Fm ratio, decreased from the second week of salinization in leaf 4 but only at the end of the third week in the younger leaf 5. In the prematurely senescent leaf 4, ABA content increased linearly while IAA strongly decreased with salinization time. Although zeatin (Z) levels were scarcely affected by salinity, zeatin-riboside (ZR) and the total cytokinin content (Z+ZR) progressively decreased by 50% from the imposition of the stress. ACC was the only hormonal compound that increased in leaf tissue coincident with the onset of oxidative damage and the decline in chlorophyll fluorescence, and prior to massive Na+ accumulation. Indeed, (Z+ZR) and ACC contents and their ratio (Z+ZR/ACC) were the hormonal parameters best correlated with the onset and progression of leaf senescence. The influence of different hormonal changes on salt-induced leaf senescence is discussed.

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Soil salinity and drought compromise water uptake and lead to osmotic adjustment in xero-halophyte plant species. These important environmental constraints may also have specific effects on plant physiology. Stress-induced accumulation of osmocompatible solutes was analysed in two Tunisian populations of the Mediteranean shrub Atriplex halimus L.-plants originating from a salt-affected coastal site (Monastir) or from a non-saline semi-arid area (Sbikha)-were exposed to nutrient solution containing either low (40 mM) or high (160 mM) doses of NaCl or 15% polyethylene glycol. The low NaCl dose stimulated plant growth in both populations. Plants from Monastir were more resistant to high salinity and exhibited a greater ability to produce glycinebetaine in response to salt stress. Conversely, plants from Sbikha were more resistant to water stress and displayed a higher rate of proline accumulation. Proline accumulated as early as 24 h after stress imposition and such accumulation was reversible. By contrast, glycinebetaine concentration culminated after 10 d of stress and did not decrease after the stress relief. The highest salt resistance of Monastir plants was not due to a lower rate of Na(+) absorption; plants from this population exhibited a higher stomatal conductance and a prodigal water-use strategy leading to lower water-use efficiency than plants from Sbikha. Exogenous application of proline (1 mM) improved the level of drought resistance in Monastir plants through a decrease in oxidative stress quantified by the malondialdehyde concentration, while the exogenous application of glycinebetaine improved the salinity resistance of Sbikha plants through a positive effect on photosystem II efficiency.

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K+ (20%) and decreases in the toxic ion Na+ (by 30%) and abscisic acid (by 20–40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.