Screening and Identification of Host Proteins Interacting with Iris lactea var. chinensis Metallothionein IlMT2a by Yeast Two-Hybrid Assay.

Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis) is a well-known cadmium (Cd)-tolerant plant and we have previously shown that the metallothionein gene, IlMT2a, of the plant may be playing a key role in conferring the Cd tolerance. In this study, we have identified several proteins interacting with the IlMT2a by screening yeast two-hybrid library constructed from cDNAs isolated from Cd-treated I. lacteal var. chinensis plants. Putative functions of these proteins include those involved in photosynthesis, ROS scavenge, nutrient transport, and transcriptional regulation, to name a few. In particular, another metallothionein, which we assigned the name of IlMT3, was identified as an interacting partner of the IlMT2a. Unlike IlMT2a, it did not provide any significant protection against Cd toxicity in transgenic Arabidopsis thaliana L. (A. thaliana). To our knowledge, this is the first time ever reporting the interaction of two metallothionein proteins in plants. Learning the biological significance of the interaction between IlMT2a and IlMT3 would be the focus of future study and would be able to provide valuable insights into the understanding plant metallothionein's diverse and complex roles in coordinating many important cellular physiologies including stress responses, gene regulations, and energy metabolisms.

Full-Length Transcriptome Sequencing and Comparative Transcriptome Analysis to Evaluate Drought and Salt Stress in Iris lactea var. chinensis.

Iris lactea var. chinensis (I. lactea var. chinensis) is a perennial herb halophyte with salt and drought tolerance. In this study, full-length transcripts of I. lactea var. chinensis were sequenced using the PacBio RSII sequencing platform. Moreover, the transcriptome was investigated under NaCl or polyethylene glycol (PEG) stress. Approximately 30.89 G subreads were generated and 31,195 unigenes were obtained by clustering the same isoforms by the PacBio RSII platform. A total of 15,466 differentially expressed genes (DEGs) were obtained under the two stresses using the Illumina platform. Among them, 9266 and 8390 DEGs were obtained under high concentrations of NaCl and PEG, respectively. In total, 3897 DEGs with the same expression pattern under the two stresses were obtained. The transcriptome expression profiles of I. lactea var. chinensis under NaCl or PEG stress obtained in this study may provide a resource for the same and different response mechanisms against different types of abiotic stress. Furthermore, the stress-related genes found in this study can provide data for future molecular breeding.

Formation of iron plaque on roots of Iris pseudacorus and its consequence for cadmium immobilization is impacted by zinc concentration.

The impact of iron plaque (IP) on bioavailability of heavy metals to plants has been well documented, but the role of zinc (Zn) in modulating the associated processes remains elusive. We took Iris pseudacorus used in wetland for remediating Cd-contaminated water as an example and systematically studied the combined influence of Cd and Zn concentration on formation of IP and its consequence for immobilization and plant uptake of Cd. The experiment was conducted in hydroponic culture and in each treatment, we measured the physiological traits, activity of antioxidant enzymes (SOD, POD, CAT), mass of the IP, as well as the Cd content in both plant tissues and IP. The results showed that increasing Cd concentration resulted in a steady reduction in IP while the impact of zinc on IP was complicated and appeared to be coupled with Cd. When the Cd concentration was low (0.5 mg L-1 measured as CdCl2 2·5H2O) increasing Zn concentration reduced IP, while when the Cd concentration was increased to 5 mg L-1 increasing zinc concentration led to an increase in IP mass first followed by a decline after Zn concentration exceeded 100 mg L-1 (measured as ZnSO4·7H2O). The change in IP as affected by Zn had a strong consequence for immobilization and plant uptake of Cd. When Cd concentration was low, the IP was comparatively abundant and hence adsorbed most Cd. In contrast, when Cd concentration was high, the IP reduced and the amount of Cd taken up by plant roots and translocated to shoots and leaves increased. Both Cd immobilization and its plant uptake were modulated by Zn concentration. At low Cd concentration the combined Cd immobilized and taken up by plant peaked when the Zn concentration was 50 mg L-1, while at high Cd concentration the combined Cd reached maxima when theZn concentration was 100 mg L-1. The activity of the antioxidant enzymes changed significantly with Zn rather than with Cd. Regardless of Cd concentration, the activity of all three antioxidant enzymes increased first with zinc concentration before declining when the Zn concentration exceeded approximately 100 mg L-1 in all treatments, comparable with the change in immobilization and plant uptake of Cd as the Zn concentration increased. SEM analysis did prove the formation and variation of IP on the root surface of Iris pseudacorus in different treatments. We also found that the plant developed a survival strategy by scarifying its leaves with high Cd content. The results presented in this paper has wide implications as it revealed that care needs to be taken in applying Zn to enhance Cd immobilization and its plant uptake as exceeding the optimal application rate might reduce remediating efficiency rather than increase it.

Boron tolerance and accumulation potential of four salt-tolerant plant species.

Boron (B) is an essential element for plants, but excess B is phytotoxic. Since excess B often occurs along with high salinity in the environment, the purposes of the experiments are to screen plants that tolerate both excess B and high salinity for the remediation of B-contaminated saline water or soils. Here we tested the capacities of B tolerance and accumulation of four salt-tolerant plant species, Tripolium pannonicum, Suaeda glauca, Iris wilsonii, and Puccinellia tenuiflora using hydroponic culture systems, and compared their potential for application in phytoremediation. The maximum B supply concentrations for the survival of T. pannonicum, S. glauca, I. wilsonii, and P. tenuiflora are 40, 250, 700, and 300 mg/L, respectively. The maximum B concentrations in the shoot tissue of these plants are 0.45, 2.48, 15.21, and 8.03 mg/g DW, and in the root are 0.23, 0.70, 6.69, and 2.63 mg/g DW, respectively. Our results suggest that S. glauca, I. wilsonii, and P. tenuiflora are capable of tolerating and accumulating high levels of B, and I. wilsonii is a most promising candidate for the remediation of B-contaminated sites. This study will provide evidence in support of our future pilot studies (e.g., constructed wetlands) on the phytoremediation of B-contaminated water and soil.

Transcriptome analysis reveals complex response of the medicinal/ornamental halophyte Iris halophila Pall. to high environmental salinity.

The remediation and subsequent use of saline-alkaline land are of great significance to ecological environment construction and sustainable agricultural development. Iris halophila Pall. is a salt-tolerant medicinal and ornamental plant, which has good application prospects in the ecological construction of saline-alkaline land; therefore, study of the molecular mechanisms of salt tolerance in I. halophila has important theoretical and practical value. To evaluate the molecular mechanism of the response of I. halophila to salt toxicity, I. halophila seedlings were treated with salt (300 mM NaCl) and subjected to deep RNA sequencing. The clean reads were obtained and assembled into 297,188 unigenes. Among them, 1120 and 100 salt-responsive genes were identified in I. halophila shoots and roots, respectively. Among them, the key flavonoid and lignin biosynthetic genes, hormone signaling genes, sodium/potassium ion transporter genes, and transcription factors were analyzed and summarized. Quantitative reverse-transcription PCR analysis strengthened the reliability of the RNA sequencing results. This work provides an overview of the transcriptomic responses to salt toxicity in I. halophila and identifies the responsive genes that may contribute to its reduced salt toxicity. These results lay an important foundation for further study of the molecular mechanisms of salt tolerance in I. halophila and related species.

Effects of microcystin-LR on the tissue growth and physiological responses of the aquatic plant Iris pseudacorus L.

The release of cyanobacterial toxins during algal bloom has adverse effects on aquatic plants and animals. This study aimed to understand the toxic effects and mechanism of microcystin-LR (MC-LR) on the seedling growth and physiological responses of Iris pseudacorus L. (calamus). After a one-month exposure experiment, the growth and development of the calamus leaves were significantly inhibited, and this inhibitory effect was verified to be concentration dependent. Furthermore, the cell membrane system was damaged, and the photosynthesis was also adversely affected by MC-LR. The relative conductivity of the leaves increased from 10.96% to 97.51%, and the total chlorophyll content decreased from 0.89 mg/g to 0.09 mg/g. Notably, the behavior of the roots in the presence of MC-LR was different from that of the leaves. The seedlings needed to absorb more nutrients to maintain the normal growth at low-toxin concentrations, but the high concentration of (over 250 µg/L) MC-LR exceeded the tolerance of plants and inhibited the growth of roots. In addition, MC-LR led to an excessive accumulation of H2O2, and the seedlings enhanced the activities of catalase, peroxidase, and superoxide dismutase to resist oxidative stress. The presence of MC-LR also affected the capacity of the plants to absorb nitrogen and phosphorus. The removal efficiency of NO3--N, the main source of nitrogen, was 63.53% in the presence of 100 µg/L MC-LR. As a result, the pH increased, and the growth of plants was indirectly inhibited. Therefore, the presence of MC-LR could affect the purification efficiency of calamus in eutrophic water. This study provides theoretical support for the selection of plants in the eutrophic water.

De novo characterization of the Iris lactea var. chinensis transcriptome and an analysis of genes under cadmium or lead exposure.

Iris lactea var. chinensis (I. lactea var. chinensis) is tolerant to accumulations of cadmium (Cd) and lead (Pb). In this study, the transcriptome of I. lactea var. chinensis was investigated under Cd or Pb stresses. Using the gene ontology database, 31,974 unigenes were classified into biological process, cellular component and molecular function. In total, 13,132 unigenes were involved in enriched Encyclopedia of Genes and Genomes (KEGG) metabolic pathways, and the expression levels of 5904 unigenes were significantly changed after exposure to Cd or Pb stresses. Of these, 974 were co-up-regulated and 1281 were co-down-regulated under the two stresses. The transcriptome expression profiles of I. lactea var. chinensis under Cd or Pb stresses obtained in this study provided a resource for identifying common mechanisms in the detoxification of different heavy metals. Furthermore, the identified unigenes may be used for the genetic breeding of heavy-metal tolerant plants.

Competition, salinity, and clonal growth in native and introduced irises.

PREMISE OF THE STUDY: Iris pseudacorus spread rapidly into North America after introduction from Europe in the 1800s and now co-occurs with native I. hexagona in freshwater Louisiana wetlands. Native irises support and interact with multiple trophic levels, whereas I. pseudacorus is classified an invasive pest because it grows aggressively, reduces biodiversity, and displaces native vegetation. Salinity levels are increasing in coastal wetlands worldwide. We examined how salt-stress affects competitive interactions between these conspecifics. METHODS: We established a three-way full-factorial common-garden experiment that included species (I. pseudacorus, I. hexagona), competition (no competition, intraspecific competition, and interspecific competition), and salinity (0, 4, 8 parts per thousand NaCl), with six replicates per treatment. KEY RESULTS: After 18 mo, Iris pseudacorus produced much more biomass than the native species did (F1, 92 = 71.5, P < 0.0001). Interspecific competition did not affect the introduced iris, but biomass of the native was strongly reduced (competition × species interaction: F2, 95 = 76.7, P = 0.002). Salinity significantly reduced biomass of both species (F2, 92 = 21.8, P < 0.0001), with no species × salinity interaction (F2, 84 = 1.85, P = 0.16). CONCLUSIONS: Our results demonstrate that salt stress strongly reduced clonal reproduction in native and introduced irises; however, the introduced iris had a competitive advantage over the native, regardless of environmental salinity levels. Based on patterns in clonal reproduction, the introduced iris could potentially threaten native iris populations. We are currently investigating seed production and mortality during competition and stress because both clonal and sexual reproduction must be considered when predicting long-term population dynamics.

Pb uptake and toxicity to Iris halophila tested on Pb mine tailing materials.

Pb tolerant mechanisms, plant physiological response and Pb sub-cellular localization in the root cells of Iris halophila were studied in sand culture and the Pb mine tailings. Results showed that the activities of superoxide dismutase (SOD) and peroxidase (POD) in the underground parts and the activity of catalase (CAT) in the aboveground and underground parts increased as Pb level was enhanced. Glutathione (GSH) and ascorbic acid (AsA) contents increased by Pb treatments. Pb deposits were found in the middle cell walls or along the inner side of epibiotic protoplasm of some cells which accumulated a large quantity of Pb and died. The dry weights (DWs) of aboveground parts under all Pb tailings treatments decreased insignificantly, while the DW of the underground parts growing in the pure Pb tailings decreased significantly. Pb, Cu, Cd, and Zn contents increased significantly as the levels of Pb tailings were enhanced and Pb contents in the aboveground and underground parts reached 64.75 and 751.75 µg/g DW, respectively, at pure Pb tailings treatment. The results indicated that I. halophila is a promising plant in the phytoremediation of Pb contaminated environment. Some antioxidant enzymes, antioxidants and compartmentalization of Pb were played major roles in Pb tolerance of I. halophila.

The inhibition and adaptability of four wetland plant species to high concentration of ammonia wastewater and nitrogen removal efficiency in constructed wetlands.

Four plant species, Typha orientalis, Scirpus validus, Canna indica and Iris tectorum were selected to assess their physiological response and effects on nitrogen and COD removal to high total ammoniacal nitrogen (TAN) in constructed wetlands. Results showed that high TAN caused decreased relative growth rate, net photosynthetic rate, and leaf transpiration. C. indica and T. orientalis showed higher TAN adaptability than S. validus and I. tectorum. Below TAN of 200 mg L(-1), growth of C. indica and T. orientalis was less affected or even stimulated at TAN range 100-200 mg L(-1). However, S. validus and I. tectorum was obviously suppressed when TAN was above 100 mg L(-1). High TAN generated obvious oxidative stress showing increased proline and malondialdehyde contents, and superoxide dismutase was inhibited. It indicated that the threshold for plant self-bioremediation against high TAN was 200 mg L(-1). What's more, planted CWs showed higher nitrogen and COD removal. Removal rate of C. indica and T. orientalis was higher than S. validus and I. tectorum.

Cadmium Toxicity and Alleviating Effects of Exogenous Salicylic Acid in Iris hexagona.

Cadmium (Cd) toxictity and possible role of salicylic acid (SA) in alleviating Cd-induced toxicity were investigated on ornamental hydrophyte Iris hexagona. Compared to the control, treatments with 100 and 500 µM Cd for 7 days significantly decreased dry weight, the contents of chlorophyll, photosynthetic parameters, and increased the content of thiobarbituric acid reactive substance. Pretreatment of the roots of I. hexagona seedlings with 1 µM SA before Cd exposure may increase dry weight, photosynthetic rate, activities of antioxidant enzymes, improve the cell ultrastructure and protect plants from Cd-induced oxidative stress damage. However, SA pretreatment had no significant effect on Cd concentrations in the leaves and roots. It is suggested that SA-induced Cd tolerances in I. hexagona are likely associated with increases in antioxidant enzyme activities and vacuolar compartmentation, rather than Cd uptake.

[hnprovement on the growth and nutrient absorption of Iris hexagona by exogenous salicylic acid under Cd stress].

The effects of exogenous salicylic acid (SA) pretreatment on the biomass, water content, photosynthesis, root vigor, Cd accumulation, nutrient absorption and antioxidant enzyme activity of Iris hexagona under Cd stress were studied by using Hoagland solution culture. The results showed that SA pretreatment promoted dry mass, root vigor and net photosynthetic rate of Cd-stressed I. hexagona seedlings. Compared with only Cd treatment, the Cd accumulation of Cd-treated plants with SA pretreatment remained unaffected, however,. SA pretreatment increased contents of N, P, S, and decreased K content. Activities of antioxidant enzymes of Cd-treated seedling root were increased under SA pretreatment. In conclusion, the improvement on growth of I. hexagona by SA pretreatment under Cd stress was attributed to improving Cd tolerance rather than reducing Cd uptake. The regulation of mineral element accumulation and the increase of antioxidant enzyme activities could be explained for Cd resistance improvement induced by salicylic acid.

Organic acids on the growth, anatomical structure, biochemical parameters and heavy metal accumulation of Iris lactea var. chinensis seedling growing in Pb mine tailings.

The effect of citric acid (CA) and ethylene diamine tetraacetic acid (EDTA) on the growth, anatomical structure, physiological responses and lead (Pb) accumulation of Iris lactea var. chinensis seedling growing in Pb mine tailings for 30 days were studied. Results showed that the dry weights (DW) of roots decreased significantly under both levels of CA. The DWs of leaves and roots treated with 2 mmol/kg EDTA decreased significantly and were 23 and 54 %, respectively, lower than those of the control. The tolerant indexes of I. lactea var. chinensis under all treatments of organic acids were lower than control. The root tip anatomical structure was little affected under the treatments of 2 mmol/kg CA and 2 mmol/kg EDTA compared with control. However, the formation of photosynthesizing cells was inhibited by the treatment of 2 mmol/kg EDTA. The concentrations of chlorophyll a, chlorophyll b and total carotenoids in the leaves treated with 2 mmol/kg EDTA significantly decreased. Higher CA level and lower EDTA level could trigger the synthesis of ascorbic acid and higher level of EDTA could trigger the synthesis of glutathione. CA and EDTA could promote Pb accumulation of I. lactea var. chinensis and Pb concentration in the leaves and roots at 2 mmol/kg EDTA treatment increased significantly and reached to 160.44 and 936.08 µg/g DW, respectively, and 1.8 and 1.6 times higher than those of the control. The results indicated that I. lactea var. chinensis could be used to remediate Pb tailing and the role of EDTA in promoting Pb accumulation was better than CA did.

Opening of Iris flowers is regulated by endogenous auxins.

Flower opening in Iris (Iris×hollandica) requires elongation of the pedicel and ovary. This moves the floral bud upwards, thereby allowing the tepals to move laterally. Flower opening is requires with elongation of the pedicel and ovary. In cv. Blue Magic, we investigated the possible role of hormones other than ethylene in pedicel and ovary elongation and flower opening. Exogenous salicylic acid (SA) and the cytokinins benzyladenine (N6-benzyladenine, BA) and zeatin did not affect opening. Jasmonic acid (JA) and abscisic acid (ABA) were slightly inhibitory, but an inhibitor of ABA synthesis (norflurazon) was without effect. Flower opening was promoted by gibberellic acid (GA(3)), but two inhibitors of gibberellin synthesis (4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate, AMO-1618; ancymidol) did not change opening. The auxins indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) strongly promoted elongation and opening. An inhibitor of auxin transport (2,3,5-triodobenzoic acid, TIBA) and an inhibitor of auxin effects [α-(p-chlorophenoxy)-isobutyric acid; PCIB] inhibited elongation and opening. The data suggest that endogenous auxins are among the regulators of the pedicel and ovary elongation and thus of flower opening in Iris.

Endogenous ethylene does not regulate opening of unstressed Iris flowers but strongly inhibits it in water-stressed flowers.

The floral buds of Iris flowers (Iris x hollandica) are enclosed by two sheath leaves. Flower opening depends on lifting the flower up to a position whereby the tepals can move laterally. This upward movement is carried out by elongation of the subtending pedicel and ovary. In the pedicels and ovaries of unstressed control flowers, the concentration of ACC (1-aminocyclopropane-1-carboxylic acid) and the rate of ethylene production increased during d 0-1 of flower opening, and then decreased. Exposure to ≥200 nL L(-1) ethylene for 24 h at 20°C inhibited elongation of the pedicel+ovary, and inhibited flower opening. However, pulsing of unstressed flowers with solutions containing inhibitors of ethylene synthesis (AOA, AVG), or an inhibitor of ethylene action (STS), did not affect pedicel+ovary elongation or flower opening. When the flowers were dehydrated for 2 d at 20°C and 60% RH, they did not open when subsequently placed in water, and showed inhibited elongation in the pedicel+ovary. This dehydration treatment resulted in elevated pedicel+ovary ACC levels and in increased ethylene production. Treatment with STS prevented the increase in ACC levels and ethylene production, overcame the effect of dehydration on elongation of the pedicel+ovary, and resulted in full flower opening. It is concluded that flower opening in unstressed Iris flowers is not regulated by endogenous ethylene. An increase in endogenous ethylene above normal levels during stress, by contrast, strongly inhibited flower opening, due to its inhibitory effect on elongation of the pedicel+ovary.

The physiological response and sub-cellular localization of lead and cadmium in Iris pseudacorus L.

The seedling development and physiological responses of Iris pseudacorus L. to Pb and Cd and their combination were studied for 28 days liquid culture and sub-cellular localization of Pb and Cd in the root tip cells treated with 2,070 mg L(-1) Pb and 1,000 mg L(-1)Cd for 16 days sand culture was evaluated. Results showed that the dry weights (DWs) of shoots and roots of I. pseudacorus were significantly decreased at 500 mg L(-1)Pb and 25 mg L(-1)Cd + 500 mg L(-1)Pb treatments and the root DWs under all treatments were significantly decreased in comparison with that of control. The concentrations of Chla in the leaves were decreased at all treatments, while, the concentrations of Chlb and total carotenoids were not significantly decreased under 25 mg L(-1)Cd and 25 mg L(-1)Cd + 500 mg L(-1)Pb treatments. The MDA and proline concentrations and POD activities in the shoots and roots were increased under treatments of 500 mg L(-1)Pb and 25 mg L(-1)Cd + 500 mg L(-1)Pb, but POD activities in the shoots and roots and MDA concentrations in the shoots were significantly decreased at 25 mg L(-1) Cd treatment. The results of sub-cellular localization of Pb and Cd showed that numerous Pb deposits were found on the inner surface of died cell walls in the cortex treated with 2,070 mg L(-1) Pb and Cd deposits were found in the cell wall treated with 1,000 mg L(-1) Cd. Pb and Cd deposits were not found in the cytoplasm. The results indicated that POD and proline showed strong beneficial properties against Pb and Cd stress and there were some mechanisms keeping most cells with normal activities in the plant from Pb toxicity by sacrificing a few cells that accumulated a large amount Pb. Sub-cellular localizations of Pb and Cd in the root tip cells of I. pseudacorus were little difference with the localizations in other species of Iris in the previous studies.

Environmental effects on the maturation of the endodermis and multiseriate exodermis of Iris germanica roots.

BACKGROUND AND AIMS: Most studies of exodermal structure and function have involved species with a uniseriate exodermis. To extend this work, the development and apoplastic permeability of Iris germanica roots with a multiseriate exodermis (MEX) were investigated. The effects of different growth conditions on MEX maturation were also tested. In addition, the exodermises of eight Iris species were observed to determine if their mature anatomy correlated with habitat. METHODS: Plants were grown in soil, hydroponics (with and without a humid air gap) or aeroponics. Roots were sectioned and stained with various dyes to detect MEX development from the root apical meristem, Casparian bands, suberin lamellae and tertiary wall thickenings. Apoplastic permeability was tested using dye (berberine) and ionic (ferric) tracers. KEY RESULTS: The root apical meristem was open and MEX development non-uniform. In soil-grown roots, the exodermis started maturing (i.e. Casparian bands and suberin lamellae were deposited) 10 mm from the tip, and two layers had matured by 70 mm. In both hydro- and aeroponically grown roots, exodermal maturation was delayed. However, in areas of roots exposed to an air gap in the hydroponic system, MEX maturation was accelerated. In contrast, maturation of the endodermis was not influenced by the growth conditions. The mature MEX had an atypical Casparian band that was continuous around the root circumference. The MEX prevented the influx and efflux of berberine, but had variable resistance to ferric ions due to their toxic effects. Iris species living in well-drained soils developed a MEX, but species in water-saturated substrates had a uniseriate exodermis and aerenchyma. CONCLUSIONS: MEX maturation was influenced by the roots' growth medium. The MEX matures very close to the root tip in soil, but much further from the tip in hydro- and aeroponic culture. The air gap accelerated maturation of the second exodermal layer. In Iris, the type of exodermis was correlated with natural habitat suggesting that a MEX may be advantageous for drought tolerance.

Delay of Iris flower senescence by protease inhibitors.

Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases. Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms. The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms. It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms.