Non-invasive assessment of the physiological role of leaf aerenchyma in Hippeastrum Herb. and its relation to plant water status.

MAIN CONCLUSION: The leaf patch clamp pressure probe combined with gas exchange measurements provides a non-invasive approach for measuring leaf aerenchyma pressure and study its physiological role in plants. The non-invasive leaf patch clamp pressure probe (LPCP) measures the output pressure, Pp, in response to the pressure applied by two magnets clamped to a leaf. In many plant species, it has been observed that the diel pattern of Pp follows the changes in the leaf turgor pressure reversely. The genus Hippeastrum comprises 143 species and many hybrids and cultivars of high economic value within Amaryllidaceae. Their leaves are characterized by the presence of aerenchyma composed of lacunae, running throughout the leaf and composing most of the mesophyll volume. In Hippeastrum, the diel changes of the LPCP output pressure are the reverse of that observed on the air pressure in the leaf aerenchyma, Pa, which depends on the changes in the leaf vapor pressure occurring during photosynthesis. A theoretical model is proposed and confirmed experimentally by LPCP and gas exchange measurements. The output pressure, Pp, in Hippeastrum can be related to the plant water status through the gas exchange processes that occur during photosynthesis. Considering the natural habitats of Hippeastrum species, these results agree with the physiological role of leaf aerenchyma in facilitating gas transport and light scattering in leaves, thus contributing to the photosynthetic efficiency of these plants under adverse environments. A second, but supplemental, interpretation of the LPCP output pressure, Pp, when applied on species in which the aerenchyma constitutes most of the mesophyll volume is presented.

Climate change, land cover change, and overharvesting threaten a widely used medicinal plant in South Africa.

Medicinal plants contribute substantially to the well-being of people in large parts of the world, providing traditional medicine and supporting livelihoods from trading plant parts, which is especially significant for women in low-income communities. However, the availability of wild medicinal plants is increasingly threatened; for example, the Natal Lily (Clivia miniata), which is one of the most widely traded plants in informal medicine markets in South Africa, lost over 40% of individuals over the last 90 years. Understanding the species' response to individual and multiple pressures is essential for prioritizing and planning conservation actions. To gain this understanding, we simulated the future range and abundance of C. miniata by coupling Species Distribution Models with a metapopulation model (RAMAS-GIS). We contrasted scenarios of climate change (RCP2.6 vs. RCP8.5), land cover change (intensification vs. expansion), and harvesting (only juveniles vs. all life-stages). All our scenarios pointed to continuing declines in suitable habitat and abundance by the 2050s. When acting independently, climate change, land cover change, and harvesting each reduced the projected abundance substantially, with land cover change causing the most pronounced declines. Harvesting individuals from all life stages affected the projected metapopulation size more negatively than extracting only juveniles. When the three pressures acted together, declines of suitable habitat and abundance accelerated but uncertainties were too large to identify whether pressures acted synergistically, additively, or antagonistically. Our results suggest that conservation should prioritize the protection of suitable habitat and ensure sustainable harvesting to support a viable metapopulation under realistic levels of climate change. Inadequate management of C. miniata populations in the wild will likely have negative consequences for the well-being of people relying on this ecosystem service, and we expect there may be comparable consequences relating to other medicinal plants in different parts of the world.

Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox.

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 µM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.

Assessment of differences in morphological and physiological leaf lodging characteristics between two cultivars of Hippeastrum rutilum.

BACKGROUND: Environmental lodging stress, which is a result of numerous factors, is characterized by uncertainty. However, several studies related to lodging in cereal crops have reported that lodging in the Hippeastrum rutilum environment is very rare. Hippeastrum rutilum is a garden flower with high ornamental value and abundant germplasm resources. Under past cultivation practices, it was found that the plant types of 'Red Lion', with red flowers, and 'Apple Blossom', with pink flowers, are quite different. The leaves of 'Red Lion' are upright, while the leaves of 'Apple Blossom' show lodging, which seriously affects its ornamental value. The aims of this study were to compare the differences between the two varieties with leaf lodging and upright leaves according to morphological and physiological attributes. In this study, karyotype analysis and phenotypic morphological and physiological characteristics were compared to explore the differences between the two plant types. RESULTS: The karyotype analysis of the two cultivars showed that their chromosome types were both tetraploid plants. The results showed that the lignin content in the leaves of 'Red Lion' was high, the cross-sectional structure of the leaf vascular bundle was more stable, and the chlorophyll content was high. In addition, significantly less energy was transferred to the electron transport chain (ETR) during the photoreaction. Similarly, the results regarding the maximum photosynthetic rate (Fv/Fm), nonphotochemical quenching (NPQ) and effective quantum yield of photosystem II photochemistry (△F/Fm') all indicated that the photosynthetic capacity of "Red Lion" was greater than that of "Apple Blossom", which was affected by leaf lodging. The size of the leaves was significantly smaller, and the leaf sag angle, leaf width, and leaf tip angle presented significantly lower values in 'Red Lion' than in 'Apple Blossom', which exhibits leaf sag. The difference in these factors may be the reason for the different phenotypes of the two cultivars. CONCLUSION: The results of this study proved that lodging affects the photosynthetic capacity of Hippeastrum rutilum and revealed some indexes that might be related to leaf lodging, laying a theoretical foundation for cultivating and improving new varieties.

Occurrence of the rare plant Sternbergia colchiciflora in an urban environment.

In this paper, we report a remarkable population of the rare plant Sternbergia colchiciflora found along a busy road section in the downtown of county seat Veszprém (W-Hungary). The population contains at least 7000 individuals and spreads across fifteen grassy traffic islands. Regarding the position of individuals, their relative distance from the road/sidewalk within the traffic islands differed significantly from a hypothetical uniform distribution, with higher number of individuals situated close to the island edges than expected by chance. Besides this rare bulbous plant, several other dry grassland specialist plant species were also found in these habitat patches, e.g., Astragalus austriacus, Festuca rupicola, Filipendula vulgaris, Muscari neglectum, Petrorhagia saxifraga, Potentilla arenaria, Ranunculus illyricus, Salvia nemorosa, Sanguisorba minor, Teucrium chamaedrys, Thesium linophyllon, Verbascum phoeniceum and Vinca herbacea. The origin of these dry grassland plants in this highly disturbed urban environment is uncertain; possibly, they were introduced ca. 15 years ago in the course of earthworks during the construction of the road. Recent management (frequent and motorized lawn mowing) seems to favor the Sternbergia colchiciflora, while the habitat is currently not directly threatened. Unfortunately, the potential for local conservation appears to be highly limited.

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self-sterility and potential for ovule discounting.

Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self-pollination. Self-sterility, where ovules that are penetrated by self-pollen tubes that do not develop into seeds, is usually considered to represent either a system of late-acting self-incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen ('prepotency'). Self-sterility associated with ovule penetration by self-pollen tubes appears to be widespread among the Amaryllidaceae. We tested for self-sterility in three Cyrtanthus species - C. contractus, C. ventricosus and C. mackenii - by means of controlled hand-pollination experiments. To determine the growth rates and frequency of ovule penetration by self- versus cross-pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross-fertilisation opportunities when ovules are disabled by self-pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self- and cross pollen. We recorded full self-sterility for C. contractus and C. ventricosus, and partial self-sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self- and cross-pollen tubes, nor in the proportions of ovules penetrated by self- and cross-pollen tubes. In this species, seed set was depressed (relative to cross-pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii. These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self-sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

Y2SK2 and SK3 type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants.

Two dehydrins from Agapanthus praecox (ApY2SK2 and ApSK3) show important protective effects under complex stresses. Both ApY2SK2 and ApSK3 contain one intron and consist of a full-length cDNA of 981 bp and 1057 bp encoding 186 and 215 amino acids, respectively. ApY2SK2 and ApSK3 transgenic Arabidopsis thaliana show reduced plasma membrane damage and ROS levels and higher antioxidant activity and photosynthesis capability under salt, osmotic, cold and drought stresses compared with the wild-type. ApY2SK2 and ApSK3 are mainly located in the cytoplasm and cell membrane, and ApY2SK2 can even localize in the nucleus. In vitro tests indicate that ApY2SK2 and ApSK3 can effectively protect enzyme activity during the freeze-thaw process, and ApY2SK2 also exhibits this function during desiccation treatment. Furthermore, ApY2SK2 and ApSK3 can significantly inhibit hydroxyl radical generation. These two dehydrins can bind metal ions with a binding affinity of Co2+> Ni2+> Cu2+> Fe3+; the binding affinity of ApSK3 is higher than that of ApY2SK2. Thus, ApY2SK2 has a better protective effect on enzyme activity, and ApSK3 has stronger metal ion binding function and effect on ROS metabolism. Moreover, plant cryopreservation evaluation tests indicate that ApY2SK2 and ApSK3 transformation can enhance the seedling survival ratio from 23% to 47% and 55%, respectively; the addition of recombinant ApY2SK2 and ApSK3 to plant vitrification solution may increase the survival ratio of wild-type A. thaliana seedlings from 24% to 50% and 46%, respectively. These findings suggest that ApY2SK2 and ApSK3 can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Identification of a Hippeastrum hybridum guanylyl cyclase responsive to wounding and pathogen infection.

Guanosine 3',5'-cyclic monophosphate (cGMP) is a critical component of many (patho)physiological processes in plants whilst guanylyl cyclases (GCs) which catalyse the formation of cGMP from GTP have remained somewhat elusive. Consequently, the two major aims are the discovery of novel guanylyl cyclases and the identification of GC/cGMP mediated processes. To identify a novel GC from Hippeastrum hybridum plant and facilitate the preparation of guanylyl cyclase in an amount sufficient for further crystallographic studies, we have constructed an overproduction system for this enzyme. This gene encodes a protein of 256 amino acids, with a calculated molecular mass of 28kD. The predicted amino acid sequence contains all the typical features and shows a high identity to other plant GCs. The GST-HpGC1 was catalytically active in Escherichia coli cells and the purified, recombinant HpGC1 was able to convert GTP to cGMP in the presence of divalent cations. The used overexpression system yields a guanylyl cyclase as 6% of the bacterial cytosolic protein. Besides the identification of HpGC1 as a guanylyl cyclase, the study has shown that the level of HpCG1 mRNA changed during stress conditions. Both mechanical damage and a Peyronellaea curtisii (=Phoma narcissi) fungi infection led to an initial decrease in the HpGC1 transcript level, followed by a substantial increase during the remainder of the 48-h test cycle. Moreover, significant changes in cyclic GMP level were observed, taking the form of oscillations. In conclusion, our data unequivocally identified the product of the HpGC1 gene as a guanylyl cyclase and demonstrates that such an overproduction system can be successfully used in enzyme synthesis. Furthermore, they indicate a link between the causing stimulus (wounding, infection) and guanylyl cyclase expression and the increase in cGMP amplitude. Therefore, it is concluded that appearance of cyclic GMP as a mediator in defense and wound-healing mechanisms provides a clue to the regulation of these processes.