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.     

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.     

Proteome analysis of leaves of the desiccation-tolerant grass, Sporobolus stapfianus, in response to dehydration

Drought and its affects on agricultural production is a serious issue facing global efforts to increase food supplies and ensure food security for the growing world population. Understanding how plants respond to dehydration is an important prerequisite for developing strategies for crop improvement in drought tolerance. This has proved to be a difficult task as all of the current research plant models do not tolerate cellular dehydration well and, like all crops, they succumb to the effects of a relatively small water deficit of −4 MPa or less. For these reasons many researchers have started to investigate the usefulness of resurrection plants, plants that can survive extremes of dehydration to the point of desiccation, to provide answers as to how plants tolerate water loss. We have chosen to investigate the leaf proteome response of the desiccation-tolerant grass Sporobolus stapfianus Gandoger to dehydration to a water content that encompasses the initiation of the cellular protection response evident in these plants. We used a combination of two-dimensional Difference Gel Electrophoresis (2D-DIGE) and liquid chromatography-tandem-mass spectrometry to compare the proteomes of young leaves from hydrated plants to those dehydrated to approximately 30% relative water content. High-resolution 2D-DIGE revealed 96 significantly different proteins and 82 of these spots yielded high-quality protein assignments by tandem-mass spectrometry. Inferences from the bioinformatic annotations of these proteins revealed the possible involvement of protein kinase-based signaling cascades and brassinosteroid involvement in the regulation of the cellular protection response. Enzymes of glycolysis, both cytoplasmic and plastidic, as well as five enzymes of the Calvin cycle increased in abundance. However, the RuBisCO large subunit and associated proteins were reduced, indicating a loss of carbon fixation but a continued need to supply the necessary carbon skeletons for the constituents involved in cell protection. Changes in abundance of several proteins that appear to have a function in chromatin structure and function indicate that these structures undergo significant changes as a result of dehydration. These observations give a unique “snap-shot” of the proteome of S. stapfianus at a critical point in the passage towards desiccation.     

Evidence for the presence of photorespiration in desiccation-sensitive leaves of the C4 'resurrection' plant Sporobolus stapfianus during dehydration stress

The possible role of photorespiration as a general stress protectionmechanism, and in C₄ plant metabolism, is controversial. Inparticular, the potential involvement of photorespiration inthe acquisition of desiccation tolerance in ‘resurrection’plants is unknown. An investigation was carried out into whetherphotorespiration is present in leaves of the C₄ resurrectionplant Sporobolus stapfianus Gandoger (Poaceae) and whether itfunctions as a mechanism of stress resistance in the desiccation-tolerantyounger leaves (YL) of this plant. It is shown that the enzymesinvolved in the photorespiratory pathway maintain their activityuntil 88% relative water content (RWC) in both YL and desiccation-sensitiveolder leaves (OL). In subsequent stages of dehydration stress,the enzymatic activity declined similarly in both YL and OL.The content of the phorespiratory metabolite, serine, and ethanolamine,a direct product of serine decarboxylation, is higher in theearly stages of dehydration (88% RWC) in OL, suggesting a transientlyenhanced photorespiratory activity in these leaves. This wasconfirmed by simultaneous gas exchange and fluorescence measurements,showing suppression of the electron transport rate in OL exposedto non-photorespiratory conditions (2% O₂) at 85% RWC. It isconcluded that a higher photorespiratory electron transportoccurs in desiccation-sensitive OL, and it is therefore proposedthat the capacity to scavenge excess electrons through photorespirationdoes not contribute to protect leaves of the desiccation-tolerantYL of S. stapfianus during the stress. Key words: Ethanolamine, glycine, photorespiratory enzymes, photosynthesis, poikilohydric plant, serine Received 5 June 2007; Revised 3 September 2007 Accepted 17 September 2007     

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.     

In situ localization of glucose and sucrose in dehydrating leaves of Sporobolus stapfianus

Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus. The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a "resurrection" plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.     

Sucrose phosphate synthase activity and the co-ordination of carbon partitioning during sucrose and amino acid accumulation in desiccation-tolerant leaf material of the C4 resurrection plant Sporobolus stapfianus during dehydration

Both sucrose and amino acids accumulate in desiccation-tolerant leaf material of the C(4) resurrection plant, Sporobolus stapfianus Gandoger (Poaceae). The present investigation was aimed at examining sucrose phosphate synthase (SPS) activity and various metabolic checkpoints involved in the co-ordination of carbon partitioning between these competing pathways during dehydration. In the initial phase of dehydration, photosynthesis and starch content declined to immeasurable levels, whilst significant increases in hexose sugars, sucrose, and amino acids were associated with concomitant significant increases in SPS and pyruvate kinase (PK) activities, and maximal activity levels of phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADH-dependent glutamate synthase (NADH-GOGAT). The next phase of dehydration was characterized by changes in metabolism coinciding with net hexose sugar phosphorylation. This phase was characterized by a further significant increase in sucrose accumulation, with increased rates of net sucrose accumulation and maximum rates of SPS activity measured under both saturating and limiting (inhibitory) conditions. SPS protein was also increased. The stronger competitive edge of SPS for carbon entering glycolysis during hexose phosphorylation was also demonstrated by the further decrease in respiration and the simultaneous, significant decline in both PEPCase and PK activities. A decreased anabolic demand for 2-oxoglutarate (2OG), which remained constant, was shown by the co-ordinated decrease in GOGAT. It is proposed that the further increase in amino acids in this phase of dehydration may be in part attributable to the breakdown of insoluble proteins.     

Identification of Glyoxalase I Sequences in Brassica oleracea and Sporobolus stapfianus: Evidence for Gene Duplication Events

Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal. A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function. These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants. Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes. Received: 10 May 1997 / Accepted: 15 October 1997     

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.     

束生刚毛藻脱水和复水过程中光合作用的变化

利用叶绿素荧光技术研究了束生刚毛藻（Cladophora fascicularis）在脱水和复水过程中光合作用的变化。自然干燥状态下,束生刚毛藻迅速脱水,干燥2．5h后,脱水达90％。脱水1．5h内。光合作用的最大量子产量Fv／Fm和最大相对电子传递速率Pm分别下降了约10％和11％;此后Fv／Fm和Pm的下降速度明显加快,脱水2．5h后,已下降到初始值的一半。脱水后半饱和光强Ik逐渐下降;初始斜率α在脱水初始阶段有轻微上升,此后逐渐下降。复水后Fv／Fm、Pm、Ik和α均迅速恢复。这表明脱水可引起束生刚毛藻光合作用的降低,但光合器官并未受到不可逆损伤,因此复水后光合活性迅速恢复。这种复水后光合能力的快速恢复,有利于束生刚毛藻适应潮间带特殊的生活环境。     

Membrane lipid composition and cellular function

Membrane fatty acid composition, phospholipid composition, and cholesterol content can be modified in many different kinds of intact mammalian cells. The modifications are extensive enough to alter membrane fluidity and affect a number of cellular functions, including carrier-mediated transport, the properties of certain membrane-bound enzymes, binding to the insulin and opiate receptors, phagocytosis, endocytosis, depolarization-dependent exocytosis, immunologic and chemotherapeutic cytotoxicity, prostaglandin production, and cell growth. The effects of lipid modification on cellular function are very complex. They often vary from one type of cell to another, and they do not exert a uniform effect on all processes in a single cell line. Therefore, it is not yet possible to make any generalizations or to predict how a given system will respond to a particular type of lipid modification. Many of the functional responses probably are caused directly by the membrane lipid structural changes, which affect either bulk lipid fluidity or specific lipid domains. The conformation or quaternary structures of certain transporters, receptors, and enzymes probably are sensitive to changes in the structure of their lipid microenvironment, leading to changes in activity. Prostaglandin production is modulated by the availability of substrate fatty acids stored in the membrane phospholipids, but the underlying chemical mechanism still involves a change in membrane lipid structure. While this is the most likely mechanism, the possibility that the membrane lipid compositional change is an independent event that occurs concurrently but is not causally related to the functional perturbations also must be considered.     

Chemical composition and ultrastructure of cellular and extracellular Moraxella glucidolytica lipopolysaccharides

A cellular (LPS I) and extracellular (LPS II) lipopolysaccharide were isolated from Moraxella glucidolytica cells grown on ethanol and from the culture fluid, respectively. Both LPS were toxic when injected to mice and chick embryos. These LPS contained glucose, galactose, glucosamine, galactosamine, 2-keto-3-deoxyoctonate and lipids. By permethylation studies, glucose was found to be linked (16) and (13) in LPS I and only (16) in LPS II. Galactose was the terminal non-reducing sugar. Branching occurred at positions 3 and 4 of galactose residues. LPS I was rich in - and -hydroxylauric and -hydroxymyristic acids and LPS II contained mainly stearic and -hydroxymyristic acids. LPS I was detoxified by mild acid and alkaline treatments. It was also dissociated by sodium deoxycholate and chromatographed on Sephadex G-75. The main fraction was reassociated by removing the surfactant by dialysis. The morphology of LPS I and LPS II was examined by electron microscopy. LPS I (original and reassociated fractions) consisted exclusively of ribbons while LPS II contained ribbons and vesicles.Non-Standard Abbreviations KDO 2-Keto-3-deoxyoctonic acids - LPS Lipopolysaccharide - NaD Sodium deoxycholate     

Unusual composition of thylakoid membranes of the resurrection plant Boea hygroscopica: Changes in lipids upon dehydration and rehydration

Boea hygroscopica is a resurrection plant that is able to pass from biosis to anabiosis and vice versa following slow dehydration, but loses this ability following a rapid water loss. Fresh leaves were detached from plants grown in well-watered conditions and subjected to either rapid or slow dehydration and rehydration. Upon rehydration only slowly dried leaves revived. Analysis of thylakoid membranes revealed a rather small amount of total lipids (1,4–2 μmol g^?1 dry weight) in comparison with other flowering plants. The main glycolipid was digalactosyldiacylglycerol (DGDG) rather than monogalactosyldiacylglycerol (MGDG) as is common in higher plants. Linoleic acid was the main fatty acid (30–40 mol% of total fatty acids), while linolenic acid was present from 14 to 26 mol%. In both the fresh and rehydrated leaves nearly all lipid components were present in similar amounts. Following dehydration the DGDG/MGDG molar ratio, which was 1.1 in control and rehydrated leaves, doubled by the end of the rapid drying period and was three times as high in slowly dried leaves. The total polar lipid/free sterol molar ratio as well as the free fatty acid level assumed the highest values in the rapidly dehydrated leaves. A shift towards the more unsaturated fatty acids was observed in all lipid classes upon dehydration irrespective of whether it was slow or rapid. Our data show only small differences between rapidly and slowly dehydrated leaves which can be correlated to the capacity of slowly dehydrated leaves to revive.     

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.     

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.