Growth inhibitor effects on protoplasmic drought tolerance and protein synthesis in leaf cells of the resurrection grass, Sporobolus stapfianus

Hydrated leaves of the resurrection grass S.stapfianus Gandoger are not desiccation tolerant, but tolerance can be induced in them by moderate to severe drought stress. When brassinolide (BR) and methyljasmonic acid (MJA) were applied separately, each improved PDT by approximately 6 MPa. Exogenous abscisic acid (ABA) improved the protoplasmic drought tolerance (PDT) of suspended cells from hydrated leaves of S. stapfianus only slightly (about 1 MPa).BR, MJA or ABA treatment of leaves on fully hydrated S. stapfianus plants induced changes in the leaf protein complement (partitioned by 2-D PAG electrophoresis), with the induction of apparently novel proteins and increased and decreased abundances of other proteins. Most of the changes that were induced by MJA differed from those produced by ABA and also by BR. Two proteins increased in abundance after treatment of leaves with MJA, BR or ABA.     

Amino acid pattern and glutamate metabolism during dehydration stress in the 'resurrection' plant Sporobolus stapfianus: a comparison between desiccation-sensitive and desiccation-tolerant leaves

The present study analyses changes in nitrogen compounds, amino acid composition, and glutamate metabolism in the resurrection plant Sporobolus stapfianus during dehydration stress. Results showed that older leaves (OL) were desiccation-sensitive whereas younger leaves (YL) were desiccation-tolerant. OL lost their soluble protein more rapidly, and to a larger extent than YL. Enzymes of primary nitrogen assimilation were affected by desiccation and the decrease in the glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent GOGAT (Fd-GOGAT, EC 1.4.7.1) activities was higher in OL than in YL, thus suggesting higher sensibility to dehydration. Moreover, YL showed higher total GS enzyme activity at the end of the dehydration stress and was shown to maintain high chloroplastic GS protein content during the entire stress period. Free amino acid content increased in both YL and OL between 88% and 6% relative water content. Interestingly, OL and YL did not accumulate the same amino acids. OL accumulated large amounts of proline and gamma-aminobutyrate whereas YL preferentially accumulated asparagine and arginine. It is concluded (i) that modifications in the nitrogen and amino acid metabolism during dehydration stress were different depending on leaf development and (ii) that proline and gamma-aminobutyrate accumulation in S. stapfianus leaves were not essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine in the YL during dehydration could be important and serve as essential nitrogen and carbon reservoirs useful during rehydration. In this context, the role of GS for asparagine accumulation in YL is discussed.     

Desiccation-tolerant Sporobolus stapfianus: lipid composition and cellular ultrastructure during dehydration and rehydration

The desiccation-tolerant plant Sporobolus stapfianus was subjectedto slow dehydration and to rehydration either as a silica gel-drieddetached leaf or as an airdried plant. In detached leaves dehydrationresulted in a lower relative water content in comparison withleaves dried on the plant. Water loss caused a reduction inchlorophyll, carotenoid and lipid contents and an increase inconjugated dienes. In detached leaves, ultrastructure was alsoaffected by dehydration, showing damaged cells with alteredchloroplasts which retained large quantities of starch and lipid-likeinclusions in the stroma. Upon rehydration a continuous degradationof the chemical composition and cell organization was observedwith a further increase in peroxidation. Leaves dehydrated onthe plant showed degradation of chlorophyll and lipids, whereascarotenoids increased and conjugated dienes decreased. Desiccationcaused a vacuolar fragmentation and a decline in starch, whereaschloroplasts underwent slight alterations. Following rewateringa full recovery of chlorophyll and lipids occurred, while carotenoidsand dienes remained constant. Starch increased in the chloroplastsand there was complete recovery of the ordered cell arrangementand chloroplast organization. Of the chloroplast polar lipids,in both sets of leaves desiccation caused a reduction only inmonogalactosyldiacylglycerol, while phospholipids showed anopposite pattern, increasing in air-dried leaves and decreasingin detached leaves. Rewatering of leaves desiccated on the plantled to a complete recovery of the lipid composition, whereasdetached leaves suffered a complete lipid degradation with theloss of polyunsaturated fatty acids. Key words: Desiccation tolerance, lipids, resurrection plants, Sporobolus stapfianus, ultrastructure     

A sister group contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus

Understanding how plants tolerate dehydration is a prerequisite for developing novel strategies for improving drought tolerance. The desiccation-tolerant (DT) Sporobolus stapfianus and the desiccation-sensitive (DS) Sporobolus pyramidalis formed a sister group contrast to reveal adaptive metabolic responses to dehydration using untargeted global metabolomic analysis. Young leaves from both grasses at full hydration or at 60% relative water content (RWC) and from S. stapfianus at lower RWCs were analyzed using liquid and gas chromatography linked to mass spectrometry or tandem mass spectrometry. Comparison of the two species in the fully hydrated state revealed intrinsic differences between the two metabolomes. S. stapfianus had higher concentrations of osmolytes, lower concentrations of metabolites associated with energy metabolism, and higher concentrations of nitrogen metabolites, suggesting that it is primed metabolically for dehydration stress. Further reduction of the leaf RWC to 60% instigated a metabolic shift in S. stapfianus toward the production of protective compounds, whereas S. pyramidalis responded differently. The metabolomes of S. stapfianus leaves below 40% RWC were strongly directed toward antioxidant production, nitrogen remobilization, ammonia detoxification, and soluble sugar production. Collectively, the metabolic profiles obtained uncovered a cascade of biochemical regulation strategies critical to the survival of S. stapfianus under desiccation.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

Comparison of sucrose metabolism during the rehydration of desiccation-tolerant and desiccation-sensitve leaf material of Sporobolus stapfianus

Sucrose accumulated during dehydration is a major potential energy source for metabolic activity during rehydration. The objective of the present study was to investigate aspects of leaf sucrose metabolism during the rehydration of desiccation-tolerant Sporobolus stapfianus Gandoger (Poaceae) over a 10-day period. Comparison was then made to sucrose metabolism during the rehydration of both desiccation-tolerant excised leaf material (dehydrated attached to the parent plant) and desiccation-sensitive leaf material (dehydrated detached from the parent plant to prevent the induction of tolerance) over a 48-h period. The pattern of sugar mobilization and glycolytic enzyme activity during the rehydration of the desiccation-tolerant excised leaves was similar to that in leaves attached to the parent plants. Significant breakdown of sucrose was not apparent in the initial phase of rehydration, suggesting the utilization of alternate substrates for respiratory activity. The desiccation-tolerant excised tissues provided a suitable control to compare the metabolism of rehydrating desiccation-sensitive material. In contrast to the tolerant tissues, sucrose breakdown in the sensitive leaves commenced immediately after watering and the accumulation in hexose sugars was inversely proportionate to the decrease in sucrose content. Hexokinase (EC 2.7.1.1), PFK (ATP phosphofructokinase, EC 2.1.7.11), aldolase (EC 4.1.2.13), enolase (EC 4.2.1.11), and PK (pyruvate kinase, EC 2.7.1.40) activity levels were significantly lower in the desiccation-sensitive material during rehydration.     

Morphological and ultrastructural aspects of dehydration and rehydration in leaves of Sporobolus stapfianus

The resurrection species Sporobolus stapfianus Gandoger has been studied by LM, TEM and SEM in order to define the leaf morphology and fine structure and to analyse the cellular changes occurring during the processes of dehydration and rehydration of the plant. Some characteristics of the fully hydrated leaf and some ultrastructural and physiological events which take place during leaf wilting are discussed in relation to their possible role in plant desiccation-tolerance.The leaves of S. stapfianus show several characteristics common among xerophytic species. In the resurrection leaf they could play a role in slowing down the drying rate, thus leaving time to activate the mechanisms protecting the cell structures against drought damage. Actually, the S. stapfianus leaves do not undergo important cellular alterations during dehydration. The chloroplasts, in particular, retain part of their photosynthetic pigments and thylakoid membranes. Upon rewatering leaf recovery is rather fast and the tissue structure and cell organization of the fully hydrated state are already regained after two days.     

The effect of dehydration and rehydration on the nitrogen content of various fractions from resurrection plants

Nitrogen contents were determined in 20 species of “resurrection plants”,i.e. plants with leaves which are able to revive from an air-dry state (viz. Boea hygroscopica, Borya nitida, Cheilanthes sieberi, Coleochloa pallidior, C. setifera, Craterostigma plantagineum, Myrothamnus flabellifolia, Oropetium capense, Pellaea calomelanos, P. falcata, P, viridis, Polypodium polypodioides, Ramondia pyrenaica, Selaginella lepidophylla, Sporobolus stapfianus, Talbotia elegans,Tripogon loliiformis, Xerophyta retinervis, X. villosa, X. viscosa), and in three desiccation sensitive species (Eragrostis tenuifolia, Selaginella kraussiana andSporobolus pyramidalis). In a preponderance of resurrection plants insoluble nitrogen content fell during dehydration of intact plants and soluble non-protein N rose. Both changes were particularly marked in species which lose chlorophyll and thylakoid structure during drying. These trends were usually only partially reversed after 24 h rehydration. Recovery of¹⁴C-leucine incorporation in rehydrating leaves was slow. Leaves of desiccation sensitive vascular plants tended on the average to lose soluble protein rather than insoluble N during drying, and tended to have higher soluble non-protein N contents than tolerant plants. However, similarity in the changes in N-contents inXerophyta villosa leaves killed by airdrying compared to leaves surviving air-drying, opposes the view that death was due to excessive loss of protein.     

Desiccation tolerance in developing soybean seeds: The role of stress proteins

The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of certain "late embryogenesis abundant" (LEA) proteins suggests that these proteins reduce desiccation-induced cellular damage. The aim of the present work was to test this hypothesis. Exogenous abscisic acid (ABA) was used to elevate the level of heal-soluble LEA-like proteins in axes from immature (30 days after flowering: mid-development) seeds of soybean ( Glycine max [L.] Merrill cv. Chippewa 64). As the LEA-like proteins accumulated in response to ABA, the leakage of all elements after desiccation and subsequent rehydration markedly declined. Both LEA-like protein accumulation and the decline in desiccation-induced electrolyte leakage were apparently dependent on the presence of ABA. Both effects of ABA were inhibited by cycloheximide. Light microscopy revealed a marked effect of the ABA on cellular integrity following desiccation. Osmotic stress also caused a decrease in desiccation-induced electrolyte leakage and stimulated the accumulation of LEA-like proteins. Our data are consistent with the hypothesis that the LEA-like proteins contribute to the increase in desiccation tolerance in response to ABA, and are consistent with a general protective role for these proteins in desiccation tolerance.     

Control of crops leaf growth by chemical and hydraulic influences

Three species of forage grasses (Festuca arundinacea, Eragrostiscurvula, Sporobolus stapfianus) commonly grown in the Mediterraneanregion were subjected to a soil drying treatment. Leaf growthrate in F. arundinacea was highly sensitive to soil drying andlow growth rates were associated with high laminar turgors.The production of ABA was stimulated by soil drying and therewas a clear relation between increasing ABA accumulation andreduction in leaf growth. Leaf growth of E. cutvula, a C⁴ warmseason grass, was relatively insensitive to soil drying whichwas not accompanied by a substantial increase in leaf ABA content.S. stapfianus, a resurrection plant, was highly sensitive todecreasing soil water availability. In these two latter species,leaf growth was substantially restricted before ABA accumulationoccurred. It is suggested that reductions in laminar turgorof E. curvula and S. stapfianus may be limiting leaf growthas soil dries. The results indicated a different mechanism ofsensing and responding to reduction in soil water availabilityfor the three species studied. The relative importance of thechemical and hydraulic control of leaf growth is discussed. Key words: Leaf growth, water relations, abscisic acid, Festuca arundinacea, Eragrostis curvula, Sporobolus stapfianus     

Contents of sugars in leaves of drying desiccation tolerant flowering plants, particularly grasses

Sugar complements were analysed in extracts from leaves of desiccation tolerant species in the angiosperm families Cyperaceae, Gesneriaceae, Liliaceae, Poaceae and Velloziaceae. Total sugar content was higher in live air-dry leaves of all desiccation tolerant species (except the grass Eragrostiella nardoides; 22 µmoles/g dw) than in the dead air-dry leaves of the desiccation sensitive grass Sporobolus pyramidalis (36 µmoles/g dw). Sucrose contents rose to high levels (40–98 µmoles/g dw) in live air-dry leaves of all species (except the grass Eragrostiella nardoides in which it rose to only 11 µmoles/g dw) to become the predominant sugar. Glucose and/or fructose contents frequently were lower after leaf drying but usually these were the sugars of next highest contents in live air-dry leaves. Contents of raffinose (that has been postulated to reduce sucrose crystallization) rose to c. 10% of sucrose contents in air-dry leaves of most desiccation tolerant species (but only c. 4% in Tripogon jacquemontii) compared with c. 2% of sucrose contents in the sensitive grass S. pyramidalis. Trehalose (a rare sugar in seed-plants) was present in all but one desiccation tolerant species (Xerophyta villosa) but only in minor amounts. The results are consistent with the views that sugars play a protective role during drying of desiccation tolerant plants in general but that other factors are also involved indesiccation tolerance, that in desiccation tolerant angiospermae sucrose is generally the predominant protective sugar and that raffinose and trehalose may supplement the role of sucrose.     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.