Desiccation-tolerant Moss Tortula Ruralis Research Articles

Molecular cloning of a stress-responsive aldehyde dehydrogenase gene ScALDH21 from the desiccation-tolerant moss Syntrichia caninervis and its responses to different stresses

Aldehyde dehydrogenases (ALDHs) are key enzymes of abiotic stress-tolerance in a variety of organisms. The ALDH gene superfamily in eukaryotes has identified 22 protein families based upon sequence identity. ALDH21 is unique to mosses and represented by a single transcript gene in the desiccation-tolerant moss Tortula ruralis. We describe the cloning and characterization of an ALDH21 homologue from Syntrichia caninervis (ScALDH21), an extremely desiccation-tolerant moss found in deserts of Central Asia. The ScALDH21 cDNA is 1,452 bp and encodes a deduced polypeptide of 483 amino acids (53 kDa), approximately 97% identical to T. ruralis ALDH21 (TrALDH21A). The ScALDH21 gene was subcloned into pET26b(+) and expressed in Escherichia coli (Rosetta) to determine the peptides function in response to desiccation and salinity. Quantitative RT-PCR was used to analyze steady-state mRNA amounts in response to Abscisic acid (ABA) and desiccation. ScALDH21 transcript levels increased significantly in response to both desiccation and ABA. In the transgenic E. coli, ScALDH21 protein could be induced under the salinity and desiccation stress and was more abundant within salt-treated gametophores relative to control tissue. The data suggest that ScALDH21 participates in the stress-resistant pathways and plays an important role in response to desiccation and salinity stresses.

Ecophysiological consequences of contrasting microenvironments on the desiccation tolerant moss Tortula ruralis.

Tortula ruralis is a homoiochlorophyllous-desiccation-tolerant (HDT) moss that retains all pigments when dehydrated and rapidly recovers physiological function upon rehydration. This moss forms extensive cover in exposed and shaded areas in the sandy semi-arid grasslands of Central Europe. We hypothesized that contrasting drying regimes between these microhabitats would affect plant N status, constraints to gas exchange and growth, as well as result in altered pigment concentrations and ratios, and photochemical light-response dynamics. Furthermore, we believed T. ruralis's HDT habit would limit its ability to acclimate to altered light environment. We found that sun plant T. ruralis had lower plant mass, as well as lower tissue N, C, total photosynthetic pigment concentrations and carbon isotope discrimination (Δ) values compared to shade plant counterparts. Carotenoid/chlorophyll ratios in sun plants were typical of high light-adapted tissue, but chlorophyll a/chlorophyll b ratios were lower, more characteristic of low light-adapted tissue. This unique combination of pigment responses was accompanied by sustained lower levels of optimal quantum efficiency of PSII (F v/F m) in sun plant T. ruralis, even during favorable diurnal conditions, and reduced engagement of energy-dependent thermal dissipation (NPQ). Reciprocal transplants of sun and shade plants showed that T. ruralis is capable of short-term adjustment to altered light level, as evidenced by increases in F v/F m, NPQ, and light-adapted PSII yield (φPSII) in transplanted sun plants, and concurrent decreases in sun-transplanted shade plants. However, the performance of transplanted sun plants remained consistently below that of undisturbed shade plants. These findings show that microenvironmental variation results in different patterns of resource acquisition in this HDT moss, and that growth in the open imparts greater desiccation tolerance, and the development of a greater standing engagement of slowly reversing photoprotective mechanisms. In contrast, prolonged activity and greater resource acquisition in shaded populations may allow T. ruralis to rapidly adjust to changes following disturbance to the plant canopy, fostering the persistence of T. ruralis in these semi-arid grasslands.

Bryophytes as experimental models for the study of environmental stress tolerance: Tortula ruralis and desiccation-tolerance in mosses

The development of a complete understanding of how plants interact with the environment at the cellular level is a crucial step in advancing our ability to unravel the complexities of plant ecology particularly with regard to the role that many of the less complex plants (i.e., algae, lichens, and bryophytes) play in plant communities and in establishing areas for colonization by their more complex brothers. One of the main barriers to the advancement of this area of plant biology has been the paucity of simple and appropriate experimental models that would enable the researcher to biochemically and genetically dissect the response of less complex plants to environmental stress. A number of bryophytes model systems have been developed and they have been powerful experimental tools for the elucidation of complex biological processes in plants. Recently there has been a resurgent interest in bryophytes as models systems due to the discovery and development of homologous recombination technologies in the moss Physcomitrella patens (Hedw.) Brach & Schimp. In this report we introduce the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaert., Meyer, and Scherb, as a model for stress tolerance mechanisms that offers a great deal of promise for advancing our efforts to understand how plants respond to and survive the severest of stressful environments. T. ruralis, a species native to Northern and Western North America, has been the most intensely studied of all bryophytes with respect to its physiological, biochemical, and cellular responses, to the severest of water stresses, desiccation. It is our hope that the research conducted using this bryophyte will lay the foundationfor not only the ecology of bryophytes and other less complex plants but also for the role of desiccation-tolerance in the evolution of land plants and the determination of mechanisms by which plant cells can withstand environmental insults. We will focus the discussion on the research we and others have conducted in an effort to understand the ability of T. ruralis to withstand the complete loss of free water from the protoplasm of its cells.

A Tortula ruralis cDNA Encoding Small-Subunit Ribosomal Protein S3a: Polysomal Retention of Transcript in Response to Desiccation and Rehydration

A Tortula ruralis cDNA Rps3a, encoding a predicted polypeptide with significant similarity to the small-subunit ribosomal protein S3a, was isolated from a desiccated gametophyte cDNA library. The deduced 248 amino acid polypeptide is approximately 28 kDa, with a predicted pl of 10.09, and shares extensive identity (> 75%) with S3a ribosomal proteins from Arabidopsis thaliana and Helianthus annuus. The deduced polypeptide (RPS3a) contains three nuclear targeting signals, one of which is unique to the bryophyte S3a homologue, and is strongly predicted by PSORT to be nuclear localized (> 97% certainty). Northern blot hybridization using total and polysomal RNA demonstrated Rsp3a is constitutively expressed in moss gametophytes during a wet/dry/wet cycle. Phylogenetic analysis of the deduced amino acid sequence demonstrates T. ruralis RPS3a is most similar to S3a ribosomal proteins from Tracheophytes. Model plants, such as Arabidopsis thaliana (Somerville & Meyerowitz 1994), have been powerful experimental tools for the elucidation of complex biological processes. However, the analysis of several important plant phenotypes, most notably the ability to tolerate extreme environmental conditions, has been difficult using these models because the traits are either not clearly expressed or entirely lacking. Bryophytes that exhibit many of these important tolerant phenotypes offer realistic models for the analysis of environmental stress-tolerance. Model bryophyte systems, such as the desiccation-tolerant moss Tortula ruralis, have proven extremely useful in the study of vegetative desiccation-tolerance and post-transcriptional gene control (Bewley 1979, 1995; Oliver & Bewley 1997; Oliver & Wood 1997; Oliver et al. 1997, 1998). Analysis of organisms from these ancient clades will provide greater insight into the stress induced cellular responses of plants (Oliver & Wood 1997) and may provide unique genetic material for the enhancement of stress-tolerance within economically important angiosperms. The desiccation-tolerance ability of T. ruralis is afforded by two integrated processes: a constitutive protection system and an active rehydration induced recovery mechanism apparently unique to ryophytes (Oliver & Bewley 1997; Oliver & Wood 1997). As such, we hypothesize that genes essential to recovery and cellular repair are prefe entially expressed upon rehydration of desiccated gametophytes, and genes that are essential to limiting cellular damage are expressed under wet, drying, and rehydration conditions (Oliver & Bewley 1997; Oliver & Wood 1997; Oliver et al. 1998; Scott & Oliver 1994). In order to gain a more complete understanding of the genes involved in desiccation-tolerance, we recently characterized 152 expressed sequence tags (ESTs) derived from polysomal RNA isolated from desiccated T. ruralis gametophytes by the single pass sequencing of randomly selected clones (Wood et al. 1999). Seven of these ESTs encode either small (S) or large (L) ribosomal proteins (r-proteins) that are key structural components of a functional ribosome and contribute to the proper and efficient translation of mRNAs (Moore 1998). Plant r-proteins are categorized using a unified system of nomenclature that is based upon rat r-protein designations (BailleySerres 1998; Wool et al. 1991). There are estimated to be 78 r-proteins in rat, all with an apparent molecular mass 8.5 (Wool et al. 1995). Plant r-proteins have simPresent address: Department of Biology, University of Akron, Akron, OH 44325-3908, U.S.A. 2 The nucleotide sequence data will appear in EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AFO 93109. 3To whom correspondence should be addressed: wood @plant.siu.edu 0007-2745/99/418-425$0.95/0 This content downloaded from 157.55.39.35 on Fri, 02 Sep 2016 04:04:49 UTC All use subject to http://about.jstor.org/terms 1999] DUFF ET AL.: TORTULA RURALIS cDNA 419 ilar biophysical properties. However, due to the lack of a systematic study within a single model plant, the number of r-proteins in angiosperms is estimated to range from 75 to 92 polypeptides (Bailley-Serres 1998). We describe the isolation and characterization of a cDNA from the bryophyte Tortula ruralis encoding a polypeptide with significant similarity to ribosomal protein S3a. The r-protein S3a has been demonstrated by cellular co-purification experiments to be associated with the 40S ribosomal subunit in Drosophila melanogaster (Reynaud et al. 1997) and similar plant cDNAs have been identified in A. thaliana (Newman et al. 1994), rice (Kidou et al. 1994) and Catharanthus roseus (Ito et al. 1991). To our knowledge, T. ruralis Rps3a is the first ribosomal protein cDNA described from a bryophyte system and as such we also present a phylogenetic analysis of the deduced amino acid

Nitrate reductase activity during desiccation and rehydration of the desiccation-tolerant moss Tortula ruralis

Water stress reduces the activity of many plant enzymes. When the stress is alleviated there is generally a delay in the recovery of metabolism to unstressed levels. The enzyme nitrate reductase (EC 1.6.6.1) declines in plants experiencing water deficits and upon the alleviation of water stress its activity recovers to control levels in 1–7 days. In desiccation-tolerant plants, metabolic recovery can occur within hours following rehydration. It has been proposed that metabolic characteristics of desiccation-tolerant plants are a source of information that can be used to improve the performance of crop plants under water deficits. In this study, the effect of desiccation and hydration on nitrate reductase activity in the desiccation-tolerant moss Tortula ruralis has been investigated. It was expected that the activity of nitrate reductase would decline during desiccation and recover rapidly following rehydration. Inclusion of nitrate in the hydration medium increased the activity in the tissue. Nitrate reductase activity increased with nitrate concentration in the bathing medium up to levels of 200 mM. The highest activity measured in rehydrated moss samples was 5.23 nkat g −1 dry weight (dw). The activity declined rapidly during dehydration and was not detectable in dried moss samples after 24 h of dehydration. The recovery of activity following rehydration was dependant on the rate of the preceding dehydration. In slowly dried moss, the activity recovered to control levels in less than 8 h while rapidly dried samples required 24 h for full recovery. Nitrite accumulated during slow dehydration but did not accumulate when desiccation was rapid. Following rehydration of slowly dried moss, the amount of nitrite declined and reached a control level within 1 h. It was proposed that nitrite accumulation might provide a source of nitrogen for metabolism during the time required for nitrate reduction to resume following rehydration. Attempts to measure immunologically reactive nitrate reductase protein levels or nitrate reductase mRNA levels using heterologous DNA probes were unsuccessful, suggesting that the Tortula nitrate reductase may be significantly different from that found in algae and higher plants.

Nitrate reductase activity during desiccation and rehydration of the desiccation tolerant moss Tortula ruralis and the leafy liverwort Porella platyphylla

Nitrate reductase (NR) activity in vivo was detected in eight of nine bryophytes investigated, at levels generally lower than those reported for nitrate-using higher plants. In three species tested, pre-treatment with 5 mol m −3 KNO3 increased NR activity by a factor of three in Tortula ruraliformis and by a factor of six in Dicranum majus and Hookeria lucens. Water stress imposed osmotically by PEG or sorbitol solutions depressed NR activity to 20% of its full-turgor value at −4.5 MPa. No significant differences in the proportional change were found between the desiccation-tolerant and non-desiccation tolerant species. The original hydration level of the tissue influenced the pattern of response during subsequent water stress. Measurements on field-collected material of Tortula ruralis indicate that continuous exposure to NO3− can result in the loss of NR inducibility. In NO3− free conditions there was some indication of reduced NR activity in bryophytes that were water stressed by air drying, but the results were variable and not statistically significant. During the first hour of rehydration of fully-desiccated material (air dry for 30 d), NR activity in Tortula ruralis declined markedly in both dark and light (more steeply in the light) while in Porella platyphylla it remained at a relatively constant low level in the light and increased in the dark. After four days of dark starvation Porella platyphylla showed reduced but measurable NR activity, which was increased by supply of exogenous sugars. The possible physiological basis of these findings is discussed.

Accumulation and polysomal recruitment of transcripts in response to desiccation and rehydration of the mossTortula ruralis

In a continuing effort to understand vegetative desiccation tolerance in poikilohydric plants we have isolated cDNA clones that represent transcripts involved in the response of the desiccation-tolerant moss Tortula ruralis to desiccation and rehydration. These clones were used in Northern analyses to determine the expression characteristics of the represented transcripts following the desiccation and rehydration of the moss gametophytes. The clones were classified into three groups; hydration, recovery and constitutive, according to their recruitment into polysomal fractions in response to the treatment

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to -20 degrees C) by (31)P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (-400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.

Plant Desiccation and Protein Synthesis

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)(-) RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)(+) RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)(-) RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)(+) RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.Poly(A)(-) RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)(+) RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)(+) RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)(+) RNA for protein synthesis. The rate of synthesis of poly(A)(+) RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)(+) RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

Stability and Synthesis of Phospholipids during Desiccation and Rehydration of a Desiccation-Tolerant and a Desiccation-Intolerant Moss

The fatty acid composition of the phospholipids from the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaertn, Meyer and Scherb and the desiccation-intolerant moss Cratoneuron filicinum has been determined. No changes in composition occur in either moss as a consequence of rapid drying, but, after slow drying, there is a decline in some unsaturated fatty acids. Upon rehydration of T. ruralis after slow drying, these acids decline further; however, within 105 minutes, they regain the same levels as those in undesiccated controls. A smaller and more transient decline occurs after rapid desiccation. Most phospholipid unsaturated fatty acids decrease during rehydration of C. filicinum, and their levels are not recovered. After both rapid and slow drying of T. ruralis, acetate and glycerol are incorporated into the phospholipid fraction, although de novo synthesis, alone, might not account for the increase in unsaturated fatty acids upon rehydration. Very little acetate or glycerol is incorporated during rehydration of C. filicinum. Loss of unsaturated fatty acids from the phospholipids of T. ruralis does not appear to be associated with increased lipoxygenase activity. Furthermore, there is little correlation between the extent of peroxidation of fatty acids due to desiccation and changes in the phospholipid fraction.

Respiration in Relation to Adenosine Triphosphate Content during Desiccation and Rehydration of a Desiccation-tolerant and a Desiccation-intolerant Moss

O(2) consumption by the desiccation-tolerant moss Tortula ruralis and the desiccation-intolerant Cratoneuron filicinum increased markedly during the latter stages of desiccation. ATP content of the mosses during desiccation was not correlated with O(2) consumption, but was influenced by the rate at which the mosses lost water. The more rapid the water loss, the more ATP that was present in the dry mosses. The pattern of O(2) consumption on rehydration also was influenced by the previous rate of desiccation. After rapid desiccation of T. ruralis O(2) consumption upon rehydration was considerably elevated, and for up to 24 hours. After very slow desiccation the elevation was small and brief. Normal O(2) consumption did not occur in C. filicinum after rapid desiccation, but did so within a few hours of rehydration after slower speeds of drying. ATP levels in T. ruralis returned to normal within 5 to 10 minutes of rehydration. In C. filicinum, increases in ATP were closely correlated with O(2) consumption. These observations are considered to be related to differential damage caused to mitochondria and to cellular integrity by different speeds of water loss. The desiccation-tolerant moss appears to be able to repair the severe damage imposed by rapid desiccation whereas the desiccation-intolerant moss cannot.