Zoran Ristic

Leaf cuticle and water loss in maize lines differing in dehydration avoidance

Summary The plant cuticle plays an important role in protecting plants against water loss. The objectives of this study were to investigate the relationships between (a) epidermal water loss and epidermal cell-wall thickness and cuticle thickness, and (b) epidermal water loss and cuticular wax quantity and composition in two lines of Zea mays L. (maize) differing in dehydration avoidance. Epidermal water loss from 16-day-old plants was determined by measuring water loss in shoots and detached leaves (leaf blades) under dark conditions. The results revealed an inverse relationship between the epidermal water loss and cell-wall and cuticle thickness. A maize line with lower rates of shoot and detached-leaf water loss, ZPBL 1304, had thicker epidermal cell wall and thicker cuticle than a maize line with higher rates of shoot and detached-leaf water loss, ZPL 389. The amount of cuticular wax did not correlate inversely with epidermal water loss, suggesting that wax quantity alone may not be important in epidermal transpiration. Water flow through the cuticle may thus be more complex than simple diffusion through a homogenous lipid layer.

Heat-stress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line

Abstract. A heat-tolerant maize (Zea mays L.) line, ZPBL 1304, synthesizes a unique set of five heat-shock polypeptides of 45 kDa. Previous studies suggested that these polypeptides might play a role in the development of thermotolerance in maize (Ristic et al., 1996, J. Plant Physiol. 149:424–432; Ristic et al., 1998, J. Plant Physiol. 153:497–505). In the present study, we isolated these polypeptides, sequenced them, and investigated their subcellular distribution and origin. Of the five polypeptides of 45 kDa, three polypeptides, including the two most abundant ones, yielded amino acid sequences similar to the chloroplast and bacterial protein synthesis elongation factor (EF-Tu). This was further confirmed using an antibody raised against maize EF-Tu, which showed a very strong reaction with the 45-kDa heat-shock protein(s). Studies on subcellular distribution and origin revealed that the 45-kDa polypeptides were localized to the chloroplasts, and were likely of nuclear origin. A full-length maize EF-Tu cDNA (Zmeftu1), previously isolated from the B73 line of maize, was used as a probe for northern blot analysis of RNA extracted from the ZPBL 1304 maize line (the nucleotide and deduced amino acid sequences of Zmeftu1 are 88% identical to the rice EF-Tu sequence). Northern blots showed a 1.85-fold increase in steady-state levels of EF-Tu mRNA during heat stress. An increase in EF-Tu transcript levels during heat stress was accompanied by increased levels of the EF-Tu protein. Isolated chloroplasts from heat-stressed plants also had higher levels of EF-Tu as compared to control chloroplasts. The maize EF-Tu polypeptides showed >80% sequence similarity with the bacterial EF-Tu, which has recently been shown to function as a molecular chaperone and to play a role in the protection of other proteins from thermal denaturation (Caldas et al., 1998, J. Biol. Chem. 273:11478–11482). It is hypothesized that chloroplast EF-Tu of the ZPBL 1304 maize line plays an important role in the development of thermotolerance.

Evidence of association between specific heat-shock protein(s) and the drought and heat tolerance phenotype in maize

Summary Although differences in heat-shock protein (HSP) patterns and differences in heritable drought and/or heat tolerance have been documented, there is no genetic evidence of association of the drought and/or heat tolerance with specific alterations in HSP expression in crop plants. In this study we tested the hypothesis that specific maize HSP(s) is(are) associated with plant ability to withstand soil drying (drought) and heat stress. We previously identified a line of maize, ZPBL 1304, which is tolerant to drought and heat stress and synthesizes a 45 ku (kDa) HSP(s), and a line of maize, ZPL 389, which is sensitive to drought and heat stress and does not synthesize the 45 ku HSP(s) (Ristic et al., 1991). We investigated possible association of the 45 ku HSP(s) of ZPBL 1304 line with the drought and heat tolerance phenotype. The two lines, ZPBL 1304 and ZPL 389, were crossed, and dehydration avoidance, damage to cellular membranes, and pattern of HSP synthesis were investigated in F2 plants after exposure to soil drying and high temperature (45°C) stress conditions. The 45 ku HSP(s) of ZPBL 1304 line was(were) associated with the drought and heat tolerance phenotype. The synthesis of 45 ku HSP(s) was observed in F2 plants that displayed an increased ability to recover from soil drying and heat stress. It is possible that either the 45 ku HSP(s) play a role in recovery from drought and heat stress or their gene(s) are in close proximity to the gene(s) which encode tolerance to drought and heat stress.

Dehydration, damage to cellular membranes, and heat-shock proteins in maize hybrids from different climates

Summary We recently described a band of HSP(s) of 45 ku (kDa) in the leaf tissue of a drought and heat-resistant maize line, ZPBL 1304 (Ristic et al., 1991). This band had not been previously described in the leaves of maize and does not appear to be common in higher plants. It is not known whether this band of HSP(s) of 45 ku is unique to the ZPBL 1304 line or if a band of HSP(s) of similar molecular weight also exist in other maize genotypes. Pioneer Hi-Bred International, Inc. has developed sixteen hybrids of maize. These hybrids have been bred for resistance to drought and heat in three, climatically different, geographical regions: southern, central, and northern United States. We hypothesize that hybrids from climatically differing environments have different abilities to withstand drought and high-temperature stress conditions and synthesize specific HSPs. The objectives of this study were to determine: 1) whether Pioneer hybrids synthesize HSPs of similar molecular mass to that of the 45 ku HSP(s) found in ZPBL 1304 maize line, 2) whether hybrids from climatically differing environments differ in their abilities to resist drought and high-temperature stress conditions, and 3) whether hybrids from climatically differing environments differ in the possible synthesis of 45 ku HSP(s). Our previous observation of the 45 ku HSP(s) was not unique to a single maize line. The 45 ku HSP(s) were synthesized in maize hybrids that have been bred for resistance to drought and heat. Southern hybrids displayed greater ability to withstand drought and heat stress conditions than central and northern hybrids. Also, southern hybrids showed greater ability to synthesize HSP(s) of 45 ku than northern hybrids. It is possible that the 45 ku HSP(s) we found play a role in the development of drought and heat resistance in maize.

Ultrastructural Evidence That Intracellular Ice Formation and Possibly Cavitation Are the Sources of Freezing Injury in Supercooling Wood Tissue of Cornus florida L

Although cellular injury in some woody plants has been correlated with freezing of supercooled water, there is no direct evidence that intracellular ice formation is responsible for the injury. In this study we tested the hypothesis that injury to xylem ray parenchyma cells in supercooling tissues is caused by intracellular ice formation. The ultrastructure of freezing-stress response in xylem ray parenchyma cells of flowering dogwood (Cornus florida L.) was determined in tissue prepared by freeze substitution. Wood tissue was collected in the winter, spring, and summer of 1992. Specimens were cooled from 0 to -60[deg]C at a rate of 5[deg]C h-1. Freezing stress did not affect the structural organization of wood tissue, but xylem ray parenchyma cells suffered severe injury in the form of intracellular ice crystals. The temperatures at which the ice crystals were first observed depended on the season in which the tissue was collected. Intracellular ice formation was observed at -20, -10, and -5[deg]C in winter, spring, and summer, respectively. Another type of freezing injury was manifested by fragmented protoplasm with indistinguishable plasma membranes and damaged cell ultrastructure but no evidence of intracellular ice. Intracellular cavitation may be a source of freezing injury in xylem ray parenchyma cells of flowering dogwood.

Response of Xylem Ray Parenchyma Cells of Red Osier Dogwood (Cornus sericea L.) to Freezing Stress (Microscopic Evidence of Protoplasm Contraction)

Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.

Response of Woody Plant Cells to Dehydrative Stress

Low temperature scanning electron microscopy was used to evaluate changes in cell and tissue morphology in response to a dehydrative stress. Bark cortical cells of both Cornus florida L. (flowering dogwood) and Cornus sericea L. (red osier dogwood) were collapsed at low tissue moisture. Bark cortical cell volume was reduced, and cell walls appeared buckled and distorted. In contrast, the shape and volume of xylem ray parenchyma cells did not appear to change in response to the dehydrative stress treatment. There was no evidence of cell wall buckling, distortion, or separation in either of the two species. Instead the xylem ray parenchyma cells within stressed tissue appeared the same as controls. These observations were similar to those reported regarding the response of woody tissues during freezing. The results do not support the hypothesis that rigid cell walls are a characteristic of plants that exhibit deep supercooling and that differences in cell wall rigidity distinguish species that supercool from species that do not.

Synthesis of a family of 45 ku heat shock proteins in a drought and heat resistant line of maize under controlled and field conditions

Summary Previous studies have shown that the leaf tissue of a drought and heat resistant line of maize ( Zea mays L.) ZPBL 1304 synthesizes unique heat shock proteins (HSPs) of 45 ku (kDa) (Ristic et al., 1991). The synthesis of 45 ku HSPs, however, has been studied only at 45 °C and in young (3-week-old) plants. In this study, we have further characterized the synthesis of 45 ku HSPs in the ZPBL 1304 line by investigating: 1) the temperature range of their synthesis, 2) the possible impact of exogenous abscisic acid (ABA) on their synthesis, 3) the possible synthesis of 45 ku HSPs in root tissue, and 4) the possible synthesis of 45 ku HSPs in mature plants (plants at the flowering stage) under field conditions. The 45 ku HSPs of the ZPBL 1304 maize line were synthesized over a broad temperature range starting from 35 °C upwards, and the synthesis of these HSPs increased with increasing temperature. Treatment with ABA did not seem to have an effect on the induction and synthesis of the 45 ku HSPs. Root tissue did not accumulate HSPs of 45 ku during heat shock. Instead, roots accumulated HSPs of 39 ku, 47ku, and 52 ku. Field-grown mature plants of the ZPBL 1304 line were found to synthesize the 45 ku HSPs. It is possible that maize HSPs of 45 ku may be important in the development of heat resistance under field conditions.

Higher Chilling Tolerance in Maize is not Always Related to the Ability for Greater and Faster Abscisic Acid Accumulation

Summary A hypothesis has been developed that in maize ( Zea mays L.) the ability for production of abscisic acid (ABA) is an important prerequisite for chilling tolerance (Capell and Dorffling, 1993). According to this hypothesis, it is expected that under low temperature conditions genotypes with higher chilling tolerance accumulate ABA faster and have higher ABA levels than genotypes with lower chilling tolerance. We tested this hypothesis by examining ABA levels and chilling tolerance in two inbred lines of maize, ZPBL 1304 and ZPL 389. Experiments were conducted under laboratory conditions. Sixteen-day-old plants were exposed to 4 °C for up to 6 d and then allowed to recover for 8 d. Chilling tolerance was assessed by examining leaf relative water content and injury to plasma and thylakoid membranes during and after a chilling treatment. ZPBL 1304 line showed a better ability to withstand chilling stress than ZPL 389. Contrary to predictions, the two maize lines displayed similar abilities to accumulate ABA and had similar levels of endogenous ABA under low temperature conditions. The results suggest that maize genotypes with higher chilling tolerance do not have a greater ability to accumulate ABA and do not have higher levels of ABA under chilling stress than genotypes with lower chilling tolerance. In some maize genotypes the ability to produce ABA may not be a prerequisite for chilling tolerance.

Response of Xylem Ray Parenchyma Cells of Supercooling Wood Tissues to Freezing Stress: Microscopic Study

Intracellular ice formation and possibly cavitation have been shown to be sources of freezing injury in ray parenchyma cells of supercooled wood tissue of flowering dogwood (Cornus florida L.). In this study we examined (1) whether the response of ray parenchyma cells to freezing stress observed in wood tissue of flowering dogwood also occurs in wood tissues of other supercooling species, and (2) whether the response of xylem ray parenchyma cells of flowering dogwood under laboratory freeze-stress conditions also occurs under field conditions. We use freeze substitution and transmission electron microscopy to study (1) the response of ray parenchyma cells of supercooled wood tissues of apple (Malus domestica Borkh) and peach (Prunus persica [L.] Batsch) to a controlled freezing stress, and (2) the response of ray parenchyma cells of wood tissue of flowering dogwood to field freezing stress. Apple and peach wood tissues were collected in winter and cooled from 0⚬ to -60⚬C at 5⚬C h-1. Flowering dogwood wood tissue was collected in winter when air temperature was -17⚬C. The responses of xylem ray parenchyma cells of apple, peach, and flowering dogwood to freezing stresses were similar. Freezing stresses did not affect the structural organization of the wood tissues. The xylem ray parenchyma cells, however, suffered two distinct types of injury. One type of injury was intracellular ice, and the other was fragmented protoplasm with indistinguishable cell ultrastructure but no evidence of intracellular ice formation. Current and previous observations supported the hypothesis that intracellular ice formation, and possibly cavitation, were responsible for freezing injury in supercooled wood tissues.