Tomato expressing Arabidopsis glutaredoxin gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components.

Chilling stress is a production constraint of tomato, a tropical origin, chilling-sensitive horticultural crop. The development of chilling tolerant tomato thus has significant potential to impact tomato production. Glutaredoxins (GRXs) are ubiquitous oxidoreductases, which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis. Here, we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development. AtGRXS17-expressing tomato plants displayed lower ion leakage, higher maximal photochemical efficiency of photosystem II (Fv/Fm) and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge. Furthermore, chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H2O2 accumulation. At the same time, temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1 (SlCBF1) and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants, and proline concentrations remained unchanged relative to wild-type plants under chilling stress. Green fluorescent protein -AtGRXS17 fusion proteins, which were initially localized in the cytoplasm, migrated into the nucleus during chilling stress, reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses. Together, our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species.

Ectopic expression of Arabidopsis glutaredoxin AtGRXS17 enhances thermotolerance in tomato.

While various signalling networks regulate plant responses to heat stress, the mechanisms regulating and unifying these diverse biological processes are largely unknown. Our previous studies indicate that the Arabidopsis monothiol glutaredoxin, AtGRXS17, is crucial for temperature-dependent postembryonic growth in Arabidopsis. In the present study, we further demonstrate that AtGRXS17 has conserved functions in anti-oxidative stress and thermotolerance in both yeast and plants. In yeast, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double-mutant cells to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm and the nuclear envelope but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. This enhanced thermotolerance correlated with increased catalase (CAT) enzyme activity and reduced H2O2 accumulation in AtGRXS17-expressing tomatoes. Furthermore, during heat stress, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing transgenic plants compared with wild-type controls. Thus, these findings suggest a specific protective role of a redox protein against temperature stress and provide a genetic engineering strategy to improve crop thermotolerance.

Environmental stresses induce health-promoting phytochemicals in lettuce.

Plants typically respond to environmental stresses by inducing antioxidants as a defense mechanism. As a number of these are also phytochemicals with health-promoting qualities in the human diet, we have used mild environmental stresses to enhance the phytochemical content of lettuce, a common leafy vegetable. Five-week-old lettuce (Lactuca sativa L.) plants grown in growth chambers were exposed to mild stresses such as heat shock (40 degrees C for 10 min), chilling (4 degrees C for 1d) or high light intensity (800 micromolm(-2)s(-1) for 1d). In response to these stresses, there was a two to threefold increase in the total phenolic content and a significant increase in the antioxidant capacity. The concentrations of two major phenolic compounds in lettuce, chicoric acid and chlorogenic acid, increased significantly in response to all the stresses. Quercetin-3-O-glucoside and luteolin-7-O-glucoside were not detected in the control plants, but showed marked accumulations following the stress treatments. The results suggest that certain phenolic compounds can be induced in lettuce by environmental stresses. Of all the stress treatments, high light produced the greatest accumulation of phenolic compounds, especially following the stress treatments during the recovery. In addition, key genes such as phenylalanine ammonia-lyase (PAL), l-galactose dehydrogenase (l-GalDH), and gamma-tocopherol methyltransferase (gamma-TMT) involved in the biosynthesis of phenolic compounds, ascorbic acid, and alpha-tocopherol, respectively, were rapidly activated by chilling stress while heat shock and high light did not appear to have an effect on the expression of PAL and gamma-TMT. However, l-GalDH was consistently activated in response to all the stresses. The results also show that these mild environmental stresses had no adverse effects on the overall growth of lettuce, suggesting that it is possible to use mild environmental stresses to successfully improve the phytochemical content and hence the health-promoting quality of lettuce with little or no adverse effect on its growth or yield.

Secondary metabolism and antioxidants are involved in environmental adaptation and stress tolerance in lettuce.

Lettuce (Lactuca sativa) plants grown in a protective environment, similar to in vitro conditions, were acclimated in a growth chamber and subjected to water stress to examine the activation of genes involved in secondary metabolism and biosynthesis of antioxidants. The expression of phenylalanine ammonia-lyase (PAL), gamma-tocopherol methyl transferase (gamma-TMT) and l-galactose dehydrogenase (l-GalDH) genes involved in the biosynthesis of phenolic compounds, alpha-tocopherol and ascorbic acid, respectively, were determined during plant adaptation. These genes were activated in tender plants, grown under protective conditions, when exposed to normal growing conditions in a growth chamber. A large increase in transcript level for PAL, a key gene in the phenylpropanoid pathway leading to the biosynthesis of a wide array of phenolics and flavonoids, was observed within 1h of exposure of tender plants to normal growing conditions. Plant growth, especially the roots, was retarded in tender plants when exposed to normal growing conditions. Furthermore, exposure of both protected and unprotected plants to water stress resulted in the activation of PAL. PAL inhibition by 2-aminoindan-2-phosphonic acid (AIP) rendered these plants more sensitive to chilling and heat shock treatments. These results suggest that activation of secondary metabolism as well as the antioxidative metabolism is an integral part of plant adaptation to normal growing conditions in lettuce plants.

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation.

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis.

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD alpha-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD alpha-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD alpha. The action of PLD alpha during freezing is of special interest because Arabidopsis plants that are deficient in PLD alpha have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD alpha-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.

Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana.

Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. The endogenous glycine betaine level in the leaves increased sharply during cold acclimation treatment as plants gained freezing tolerance. When glycine betaine (10 mM) was applied exogenously to the plants as a foliar spray, the freezing tolerance increased from -3.1 to -4.5 degrees C. In addition, when ABA (1 mM) was applied exogenously, the endogenous glycine betaine level and the freezing tolerance in the leaves increased. However, the increase in the leaf glycine betaine level induced by ABA was only about half of that by the cold acclimation treatment. Furthermore, when plants were subjected to water stress (leaf water potential of approximately -1.6 MPa), the endogenous leaf glycine betaine level increased by about 18-fold over that in the control plants. Water stress lead to significant increase in the freezing tolerance, which was slightly less than that induced by the cold acclimation treatment. The results suggest that glycine betaine is involved in the induction of freezing tolerance in response to cold acclimation, ABA, and water stress in Arabidopsis plants.

Freezing Characteristics of Rigid Plant Tissues (Development of Cell Tension during Extracellular Freezing).

The freezing characteristics and development of cell tension during extracellular freezing were examined in supercooling stem tissues of riverbank grapes (Vitis riparia) and cold-hardened leaves of live oak (Quercus virginiana) and mountain cranberry (Vaccinium vitis-idaea). Dormant stem xylem and pith tissues of river-bank grapes were resistant to freeze-induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced cell tension in the leaves of live oak and mountain cranberry. Maximum cell tension in the leaves of live oak was 16.8 MPa and 8.3 MPa in the leaves of mountain cranberry. Following peak tensions in the leaves, a decline in the pressure was observed with progressive freezing. The results suggest that resistance to cell deformation during extracellular freezing due to cell-wall rigidity can lead to reduced cell dehydration and increased cell tension. A relationship to predict freezing behavior in plant tissues based on cell rigidity is presented. Based on cell-water relations and ice nucleation rates, cell-wall rigidity has been shown to effect the freezing characteristics of plant tissues, including freeze-induced dehydration, supercooling, and homogeneous nucleation temperatures.

Cell-Wall Changes and Cell Tension in Response to Cold Acclimation and Exogenous Abscisic Acid in Leaves and Cell Cultures.

Freeze-induced cell tensions were determined by cell water relations in leaves of broadleaf evergreen species and cell cultures of grapes (Vitis spp.) and apple (Malus domestica). Cell tensions increased in response to cold acclimation in leaves of broadleaf evergreen species during extracellular freezing, indicating a higher resistance to cell volume changes during freezing in cold-hardened leaves than in unhardened leaves. Unhardened leaves, typically, did not develop tension greater than 3.67 MPa, whereas cold-hardened leaves attained tensions up to 12 MPa. With further freezing there was a rapid decline and a loss of tension in unhardened leaves of all the broadleaf evergreen species studied. Also, similar results were observed in cold-hardened leaves of all of the species except in those of inkberry (Ilex glabra) and Euonymus fortunei, in which negative pressures persisted below -40[deg]C. Abscisic acid treatment of inkberry and Euonymus kiautschovica resulted in increases in freeze-induced tensions in leaves, suggesting that both cold acclimation and abscisic acid have similar effects on freezing behavior[mdash] specifically on the ability of cell walls to undergo deformation. Decreases in peak tensions were generally associated with lethal freezing injury and may suggest cavitation of cellular water. However, in suspension-cultured cells of grapes and apple, no cell tension was observed during freezing. Cold acclimation of these cells resulted in an increase in the cell-wall strength and a decrease in the limiting cell-wall pore size from 35 to 22 A in grape cells and from 29 to 22 A in apple cells.

Characterization of heat injury in grapes using h nuclear magnetic resonance methods : changes in transverse relaxation times.

Transverse relaxation times (T(2)) of tissue water ((1)H) in leaves and suspension cultured cells of grape hybrids (Vitis spp. cv ;Venus' and ;Veeblanc') were measured by nuclear magnetic resonance at various temperatures. The tissue water was characterized by two T(2) time constants. A sharp decrease in T(2) for the major fraction of tissue water was observed in association with heat injury, as measured by electrolyte leakage and triphenyltetrazolium chloride reduction in both leaves and suspension cultured cells. The changes in T(2) as a result of heat injury were irreversible, as indicated by a temperature dependent hysteresis of T(2). Studies using a paramagnetic probe (Mn(+2)) indicated that the plasma membrane was irreversibly damaged at the killing temperature, resulting in a loss of cell compartmentalization. Tissue water in heat-killed samples was characterized by only a single T(2).

Supercooling characteristics of isolated peach flower bud primordia.

The amount of unfrozen water in dormant peach (Prunus persica [L.] Batsch, cv Redhaven) flower buds, isolated primordia, and bud axes was determined during freezing using pulse nuclear magnetic resonance methods. Differential thermal analysis studies were conducted on whole buds and isolated primordia in the presence of ice nucleation. The results showed that some of the water in isolated primordia remained supercooled in the presence of ice nucleation. Although most tissue water froze (57.5%) following ice nucleation at -2.5 degrees C, a considerable amount of water was found to supercool. In the presence of ice nucleation, increased hydration of isolated primordia resulted in the elimination of the supercooling characteristic. The structural integrity of isolated primordia appeared to be essential for supercooling.

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants.

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0 degrees C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between -3.5 degrees and -8.5 degrees C. However, if ice initiation in the supercooled leaves occurred at any temperature below -2.6 degrees C, the leaves were lethally injured.To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above -1 degrees C. They had threshold temperatures of -0.7 degrees and -0.8 degrees C, respectively, for freezing in distilled H(2)O. Although freezing could be initiated in the Solanum leaves above -1 degrees C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between -2.5 degrees and -6.5 degrees using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.