Marked differences in seed dormancy in two populations of the Mediterranean shrub,Cistus albidus L.

Background: Population structure and dynamics in natural ecosystems can be affected by seed viability and dormancy. However, how the endogenous contents of phytohormones and vitamin E in seeds relative to the environment affect viability and dormancy is not yet fully understood.

Aims: We studied seed viability and germination capacity in two populations of the Mediterranean shrub, Cistus albidus, exposed to contrasting environmental conditions.

Methods: We measured seed viability and germination capacity and endogenous contents of abscisic acid (ABA), gibberellins and vitamin E in seeds collected from two populations, in two environmentally contrasting environments in north-eastern Spain.

Results: Plants growing in the natural site produced seeds with lower germination percentage but similar viability, indicating higher seed dormancy. Enhanced seed dormancy was paralleled with higher contents of ABA and lower contents of gibberellins (GAs). Contents of tocopherols and tocotrienols were higher in seeds of the natural population. Tocopherol contents in seeds correlated positively with the ratio living:total aerial biomass.

Conclusions: Two Mediterranean populations of C. albidus growing in two sites with contrasting environmental conditions showed marked differences in seed dormancy and germination, which may be explained, at least in part, by differences in seed hormonal contents.     

Loss of flower bud vigour in the Mediterranean shrub,Cistus albidus L. at advanced developmental stages

To better understand aging in perennials, age‐related changes in the physiology of leaves and flower buds of the Mediterranean shrub, Cistus albidus L. were evaluated. Two groups of different ages (5 and 10 years old), both at advanced developmental stages but of similar size, were compared. Total plant biomass, biomass produced per apical meristem and levels of cytokinins, abscisic acid and jasmonic acid in leaves and flower buds, as well as flower production, were measured. No differences in plant size, vegetative growth rates and levels of phytohormones in leaves were observed between 5‐ and 10‐year‐old plants. However, they showed significant differences in flower bud development; the older plants having reduced vigour, with 29.6% of flowers reaching anthesis compared to 52.5% in the younger plants. Furthermore, endogenous concentrations of zeatin and abscisic acid in flower buds at stage I (start of flower organ formation) were 61% and 41%, respectively, smaller in 10‐ than in 5‐year‐old plants. At stage II (with all flower organs formed), zeatin and abscisic acid concentrations decreased by ca. 90% and 80%, respectively, but differences between age groups were still evident (60% and 29% for zeatin and abscisic acid, respectively). Jasmonic acid levels in flower buds decreased by 80% from stage I to II, but did not differ between age groups. Despite reductions in flower bud vigour, total number of flowers per individual was not significantly different between age groups, so that an age‐related loss in reproductive vigour at the organ level did not lead to a decrease in flower production at the whole plant level.     

Climatic and fire determinants of early life-history stages in the Mediterranean shrub Cistus albidus

在伊比利亚半岛东北部沿水分梯度的典型地中海灌丛中对白毛岩蔷薇（Cistus albidus）的繁殖性状进行研究。火对该物种的萌发非常有利。此外,地中海的物种特别依赖于水分有效性。因此,我们建立了一个假说,即除火灾干扰外,该地中海植物的幼苗更新将因干旱事件得到改善,主要通过干旱事件引发冠层大量死亡。本研究收集了白毛岩蔷薇几个种群的个体,并对果实和种子的大小、重量和数量进行测定。同时也对五种发芽前处理进行了发芽测试：热激、吸胀、两个吸胀/干燥循环,以及热激和吸胀与吸胀/干燥循环的组合。此外,在野外对干旱事件后的幼苗数量进行了调查,并与冠层死亡进行相关分析。我们的研究表明,沿水分利用梯度,白毛岩蔷薇繁殖性状（例如发芽率和果实产量）具有变异性。这种变异性导致干旱条件下果实产量减少而发芽率增加。白毛岩蔷薇种子会因受热增加发芽率,表明它们具有在火灾后能够成功更新的能力。然而,白毛岩蔷薇种群的增长并不完全取决于火,因为在极端干旱后死亡的白毛岩蔷薇冠层下的幼苗更新率更高。最后,成年个体密度增加了白毛岩蔷薇的死亡以及幼苗的更新。这些结果表明,该物种在不同的繁殖结果（即种子产量与存活率）间表现出权衡,而这种权衡又取决于气候。这项研究也为种内竞争、气候（尤其是干旱事件和火干扰）如何影响常见且具有代表性的地中海易火灌木的生活史关键早期阶段的成功提供了证据。     

Earlier summer drought affects leaf functioning of the Mediterranean species Cistus monspeliensis L.

The Mediterranean vegetation is characterized by a high diversity of growth forms, habits and phenology that enable it to endure under harsh environmental conditions. It is however unclear whether these adaptations may allow plant survival under more extreme conditions, as predicted by climatic models under the perspective of climate change. A manipulative experiment aiming at anticipating summer aridity has been run to analyse the effects of the experimental drought on spring-leaf functioning and characteristics of the leaf-dimorphic Mediterranean shrub Cistus monspeliensis L.Assimilation rates were reduced under anticipated summer aridity due to a decrease of stomatal conductance, but only before morphological adaptations to drought (an increase of leaf mass per area) occurred. These adaptations were anticipated under experimental dry conditions, and causes photosynthetic performances to recover compared to previous dates. When natural summer aridity occurred, the leaf mass per area also changed in the control. However, this causes no recovery of the photosynthetic performances, because of the decrease of stomatal conductance due to low soil water content and leaf water potential values. Moreover, under experimental drought, leaf shedding was anticipated to reduce water losses, causing an overall reduction of leaf lifespan.     

The Mediterranean evergreen Quercus ilex and the semi-deciduous Cistus albidus differ in their leaf gas exchange regulation and acclimation to repeated drought and re-watering cycles

Plants may exhibit some degree of acclimation after experiencing drought, but physiological adjustments to consecutive cycles of drought and re-watering (recovery) have scarcely been studied. The Mediterranean evergreen holm oak (Q. ilex) and the semi-deciduous rockrose (C. albidus) showed some degree of acclimation after the first of three drought cycles (S1, S2, and S3). For instance, during S2 and S3 both species retained higher relative leaf water contents than during S1, despite reaching similar leaf water potentials. However, both species showed remarkable differences in their photosynthetic acclimation to repeated drought cycles. Both species decreased photosynthesis to a similar extent during the three cycles (20-40% of control values). However, after S1 and S2, photosynthesis recovered only to 80% of control values in holm oak, due to persistently low stomatal (g(s)) and mesophyll (g(m)) conductances to CO(2). Moreover, leaf intrinsic water use efficiency (WUE) was kept almost constant in this species during the entire experiment. By contrast, photosynthesis of rockrose recovered almost completely after each drought cycle (90-100% of control values), while the WUE was largely and permanently increased (by 50-150%, depending on the day) after S1. This was due to a regulation which consisted in keeping g(s) low (recovering to 50-60% of control values after re-watering) while maintaining a high g(m) (even exceeding control values during re-watering). While the mechanisms to achieve such particular regulation of water and CO(2) diffusion in leaves are unknown, it clearly represents a unique acclimation feature of this species after a drought cycle, which allows it a much better performance during successive drought events. Thus, differences in the photosynthetic acclimation to repeated drought cycles can have important consequences on the relative fitness of different Mediterranean species or growth forms within the frame of climate change scenarios.     

Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins

Cistus ladanifer L. (Cistaceae) is a Mediterranean shrub covering different kinds of soils in the Western Mediterranean area. This species has colonised several metalliferous areas (serpentine outcrops as well as human-polluted sites) throughout its distribution range, and is therefore an interesting species to study the possible effects on genetic diversity and differentiation produced by the colonisation of areas polluted with heavy metals. The genetic structure of 33 natural populations distributed across its entire natural distribution range (Morocco, Portugal and Spain) and growing on either metalliferous or non-metalliferous soils was investigated using chloroplast microsatellites. Population genetic parameters were estimated and genetic groups were identified using Bayesian inference. In addition, we compared the genetic diversity and differentiation among metallicolous and non-metallicolous populations within each Bayesian-defined group. The cpSSR data suggested that metallicolous populations of Cistus ladanifer have arisen through multiple independent evolutionary origins within two different chloroplast lineages. Evidence that the soil type provoked genetic bottlenecks in metallicolous populations or genetic differentiation among metallicolous and non-metallicolous populations was not observed. Historical factors are the main cause of the present genetic structure of C. ladanifer. The nature of tolerance to heavy metals as a species-wide trait in this shrub is discussed.     

Hydrogen peroxide is involved in the regulation of rice (Oryza sativa L.) tolerance to salt stress

In the present study, we investigated the salt tolerance mechanism of two rice cultivars (Zhenghan-2 and Yujing-6), which show different tolerance to drought and disease. NaCl induced higher extent of lipid peroxide and ion leakage in Yujing-6 roots than those in Zhenghan-2 roots. H₂O₂ accumulation in Zhenghan-2 roots was lower than that in Yujing-6 roots under salt stress. Comparatively, NaCl treatment did not increase O₂ ^? contents in both rice roots, however, O₂ ^? level in Yujing-6 roots was higher than that in Zhenghan-2 roots under both control and salt stress conditions. Ascorbate peroxidases (APX) activity increased more significantly in Zhenghan-2 roots than that in Yujing-6 roots. The activity of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase (G6PDH) was similarly enhanced in both rice roots under salt stress; however, they showed higher levels in Zhenghan-2 roots than in Yujing-6 roots. Exogenous H₂O₂ could enhance APX, CAT, POD, SOD and G6PDH activities in a concentration-dependent manner in both rice roots. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted the NaCl-induced H₂O₂ accumulation, markedly decreased the activity of above enzymes. Moreover, ion leakage increased dramatically in Zhenghan-2 roots and reached to the similar level of Yujing-6 roots under NaCl+DPI treatment. Taken together, H₂O₂, which is mainly generated from PM NADPH oxidase, is involved in Zhenghan-2 rice tolerance to salt stress by enhancing the cellular antioxidant level.     

Meristem aging is not responsible for age-related changes in growth and abscisic acid levels in the Mediterranean shrub, Cistus clusii

To obtain new insights into the mechanisms underlying aging in perennials, we measured abscisic acid levels, growth and other stress indicators in leaves of Cistus clusii Dunal plants of different ages grown under Mediterranean field conditions. Recently emerged leaves from 9-year-old plants were compared to those of 1-year-old plants (obtained from cuttings from 9-year-old plants) to evaluate the effects of meristem aging on plant aging. Rooting and successful establishment of the cuttings allowed us to compare the physiology of plants with old meristems, but of different size. Plants obtained from cuttings were rejuvenated, with new leaves displaying a higher leaf area and chlorophyll content, but smaller leaf mass per unit area ratios and endogenous abscisic acid levels than those of 9-year-old plants. A comparative study in 1-, 4- and 9-year-old plants revealed that abscisic acid levels increase during the early stages of plant life (with increases of 90% between 1- and 4-year-old plants), but then remain constant at advanced developmental stages (between 4- and 9-year-old plants). Although leaf biomass was 53% smaller in 9-year-old compared to 4-year-old plants, the dry matter produced per shoot apical meristem was equivalent in both plant groups due to an increased number of leaves per shoot in the former. It is concluded that (i) C. clusii plants maintain the capacity to rejuvenate for several years; (ii) newly emerged leaves accumulate higher amounts of abscisic acid during early stages of plant life, but the levels of this phytohormone later remain constant; and (iii) although plant aging leads to the production of smaller leaves, the amount of biomass produced per shoot apical meristem remains constant at advanced developmental stages.     

Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity=0.32). High differentiation among populations was estimated (G(ST)=0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.     

Hydrogen peroxide is involved in hamster sperm capacitation in vitro

We have investigated the possibility that the generation of hydrogen peroxide (H2O2) by spermatozoa plays a physiological role during capacitation. Capacitation is defined as the incubation period required for fertilization in mammals. Capacitation culminates in an exocytotic event, the acrosome reaction (AR). Mammalian sperm generate H2O2 during aerobic incubation and do not contain catalase, the enzyme that promotes scavenging of H2O2. In the present work we show that added catalase inhibited the AR, while glucose oxidase (GO), an enzyme that generates H2O2, accelerated the onset of the AR. Direct addition of H2O2 also stimulated the AR; catalase inhibited both the stimulation by GO and by H2O2. The onset of the AR was always preceded by the appearance of hyperactivated motility. The stimulation of the AR by H2O2 was manifest 1-2 h after the addition of H2O2. Catalase added at 3 h of incubation was less effective in inhibiting the AR than catalase added at the beginning. Incubation of sperm with catalase prevented the induction of the AR by the membrane-perturbing lipid, lysophosphatidyl choline. Taken together, these results suggest that H2O2 produced by hamster sperm plays a significant role during capacitation, possibly in membrane reorganization to facilitate the fusion that takes place during exocytosis of the acrosomal contents.     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

Comparative photochemical and phenomorphological responses to winter stress of an evergreen (Quercus ilex L.) and a semi-deciduous (Cistus albidus L.) Mediterranean woody species

The impact of winter stress on plants from a Mediterranean area was evaluated through comparison of photosystem II (PS II) efficiencies and phenomorphological characteristics of two Mediterranean woody species – Quercus ilex (evergreen) and Cistus albidus (semi-deciduous). The studies were carried out in NE Spain, at two sites with different mean winter temperatures. The results showed that reductions of the efficiency of PS II may occur in Mediterranean plant communities during winter, and are especially remarkable on colder days. The extent and duration of the decrease in PS II efficiency (photoinhibition) depended not only on the climatic conditions, but also on the site, on the species considered and on the position of leaves in the plant canopy. Increased photoinhibition at the colder site was only clear for C. albidus. Nevertheless, the efficiencies of PS II were always higher in C. albidus than in Q. ilex. Phenological patterns, morphology and leaf inclination may protect C. albidus leaves from potentially photoinhibitory conditions in winter. Morphological and structural photoprotection is apparently not so well developed in Q. ilex, which probably relies more on increased protection at the photochemical level of its long-lived leaves. As has been reported in relation to summer-drought stress, Q. ilex has possibly developed a strategy of tolerance to photoinhibition, whereas C. albidus relies preferentially on avoidance features.     

Enhanced UV-B radiation increases the reproductive effort in the Mediterranean shrub Cistus creticus under field conditions

Seedlings of the Mediterranean shrub Cistus creticus L. were grown in the field under ambient or ambient plus supplemental UV-B radiation (simulating a 15% ozone depletion over Patras, 38.3°W, 29.1°E) for 20 months. During this period, measurements of photosynthetic capacity, photochemical efficiency of PS II, chlorophylls and carotenoids were performed once per season. Supplemental UV-B radiation had no significant effect on these parameters nor on the total, above ground biomass accumulation, plant height and leaf specific mass measured at plant harvest. It was observed, however, that UV-B supplementation increased the number of seeds per fruit as well as mean individual seed mass. As a result, seed number and total seed mass per plant were considerably increased. Germination rates of produced seeds were not affected. We may conclude that C. creticus is a UV-B resistant plant whose competitive ability may be improved by enhanced UV-B radiation through an increase in its reproductive effort and a higher contribution to the seed bank.     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Seasonal Dimorphism in the Mediterranean Cistus incanus L. subsp. incanus

Mediterranean perennial species are described as being sclerophyllous,or summer deciduous, or seasonally dimorphic. Field observationin the coastal maquis of Castelvolturno Nature Reserve, southernItaly, showed thatCistus incanus L. subsp. incanus is a seasonallydimorphic species as it develops brachyblasts with small leavesin summer, and dolichoblasts with large leaves in winter. Fieldbiometric data confirmed that winter shoots were 14-times longerthan those developed in summer and had many more leaves. Thearea of single winter leaves was five-times that of summer leaves.Anatomical leaf structure also changed with the season: winterleaves were flat while summer leaves had a crimped lamina whichwas partially rolled to form crypts in the lower surface. Leaveswere covered by considerably more trichomes in summer than inwinter. Stomata were uniformly distributed along the lower epidermisof winter leaves but were only present in the crypts of summerleaves. In summer leaves, a palisade layer was often found onboth sides of the lamina, the mesophyll cells were generallysmaller and the intercellular spaces were reduced. Winter leaveshad a dorsiventral structure and larger intercellular spaces.Seasonal dimorphism is generally reported to be an adaptationto summer drought. However, the morphology and anatomy of C.incanus L. subsp.incanus showed that the subspecies has notonly developed a strategy to survive summer drought, but hasevolved two different habits, one more xerophytic than the other,to optimize adaptation to the seasonal climatic changes occurringin Mediterranean environments. Copyright 2001 Annals of BotanyCompany Cistus, Cistus incanus L. subsp. incanus, climatic changes, leaf anatomy, leaf dimorphism, Mediterranean shrubs, phenology, seasonal dimorphism     

Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis

One of the most important functions of blue light (BL) is to induce chloroplast movements in order to reduce the damage to the photosynthetic machinery under excess light. Hydrogen peroxide (H(2)O(2)), which is commonly generated under various environmental stimuli, can act as a signalling molecule that regulates a number of developmental processes and stress responses. To investigate whether H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements, a laser scanning confocal microscope and a luminescence spectrometer were used to observe H(2)O(2) generation in situ with the assistance of the fluorescence probe dichlorofluorescein diacetate (H(2)DCF-DA). After treatment with high-fluence BL, an enhanced accumulation of H(2)O(2), indicated by the fluorescence intensity of DCF, can be observed in leaf cells of Arabidopsis thaliana. Exogenously applied H(2)O(2) promotes the high-fluence BL-induced chloroplast movements in a concentration-dependent manner within the range of 0-10(-4) M, not only increasing the degree of movements but also accelerating the start of migrations. Moreover, the high-fluence BL-induced H(2)O(2) generation and the subsequent chloroplast movements can be largely abolished by the administration of the H(2)O(2)-specific scavenger catalase and other antioxidants. In addition, in-depth subcellular experiments indicated that high-fluence BL-induced H(2)O(2) generation can be partly abolished by the addition of diphenyleneiodonium (DPI), which is an NADPH oxidase inhibitor, and the blocker of electron transport chain dichlorophenyl dimethylurea (DCMU), respectively. The results presented here suggest that high-fluence BL can induce H(2)O(2) generation at both the plasma membrane and the chloroplast, and that the production of H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements.