Comparison of plant biostimulating properties of Chlorella sorokiniana biomass produced in batch and semi-continuous systems supplemented with pig manure or acetate.

Microalgae-derived biostimulants provide an eco-friendly biotechnology for improving crop productivity. The strategy of circular economy includes reducing biomass production costs of new and robust microalgae strains grown in nutrient-rich wastewater and mixotrophic culture where media is enriched with organic carbon. In this study, Chlorella sorokiniana was grown in 100 l bioreactors under sub-optimal conditions in a greenhouse. A combination of batch and semi-continuous cultivation was used to investigate the growth, plant hormone and biostimulating effect of biomass grown in diluted pig manure and in nutrient medium supplemented with Na-acetate. C. sorokiniana tolerated the low light (sum of PAR 0.99 ± 0.18 mol/photons/(m2/day)) and temperature (3.7-23.7° C) conditions to maintain a positive growth rate and daily biomass productivity (up to 149 mg/l/day and 69 mg/l/day dry matter production in pig manure and Na-acetate supplemented cultures respectively). The protein and lipid content was significantly higher in the biomass generated in batch culture and dilute pig manure (1.4x higher protein and 2x higher lipid) compared to the Na-acetate enriched culture. Auxins indole-3-acetic acid (IAA) and 2-oxindole-3-acetic acid (oxIAA) and salicylic acid (SA) were present in the biomass with significantly higher auxin content in the biomass generated using pig manure (> 350 pmol/g DW IAA and > 84 pmol/g DW oxIAA) compared to cultures enriched with Na-acetate and batch cultures (< 200 pmol/g DW IAA and < 27 pmol/g DW oxIAA). No abscisic acid and jasmonates were detected. All samples had plant biostimulating activity measured in the mungbean rooting bioassay with the Na-acetate supplemented biomass eliciting higher rooting activity (equivalent to 1-2 mg/l IBA) compared to the pig manure (equivalent to 0.5-1 mg/l IBA) and batch culture (equivalent to water control) generated biomass. Thus C. sorokiniana MACC-728 is a robust new strain for biotechnology, tolerating low light and temperature conditions. The strain can adapt to alternative nutrient (pig manure) and carbon (acetate) sources with the generated biomass having a high auxin concentration and plant biostimulating activity detected with the mungbean rooting bioassay.

Growth-Promoting Characteristics of Fungal and Bacterial Endophytes Isolated from a Drought-Tolerant Mint Species Endostemon obtusifolius (E. Mey. ex Benth.) N. E. Br.

Endophytes are primarily endosymbiotic bacteria and fungi that colonize the interior tissues of their host plant. They enhance the host plant's growth and attenuate adverse effects of biological stress. Endophytic species of many indigenous plants are an untapped resource of plant growth-promoting microorganisms that can mitigate abiotic stress effects. Thus, this study aimed to isolate endophytes from the roots and leaves of the medicinal plant Endostemon obtusifolius to evaluate their in vitro growth-promoting capacities and drought tolerance and to characterize the most promising species. Twenty-six endophytes (fourteen bacteria and twelve fungi) were isolated and cultured from the roots and leaves of E. obtusifolius. All 26 endophytes produced flavonoids, and 14 strains produced phenolic compounds. Of the 11 strains that displayed good free radical scavenging capability (low IC50) in the 1-1-diphenyl-1-picryhydrazyl radical scavenging assay, only three strains could not survive the highest drought stress treatment (40% polyethylene glycol). These 11 strains were all positive for ammonia and siderophore production and only one strain failed to produce hydrogen cyanide and solubilize phosphate. Seven isolates showed aminocyclopropane-1-carboxylate deaminase activity and differentially synthesized indole-3-acetic acid. Using molecular tools, two promising symbiotic, drought stress tolerant, and plant growth-enhancing endophytic species (EORB-2 and EOLF-5) were identified as Paenibacillus polymyxa and Fusarium oxysporum. The results of this study demonstrate that P. polymyxa and F. oxysporum should be further investigated for their drought stress mitigation and plant growth enhancement effects as they have the potential to be developed for use in sustainable agricultural practices.

Bioprospecting for bioactive compounds in microalgae: Antimicrobial compounds.

While there are many opportunities to use microalgae as antimicrobial agents, little has been done to develop them beyond the characterization phase to the biotechnology phase. One challenge when screening microalgae for antimicrobial activity is their ability to synthesize biologically active secondary metabolites in response to environmental triggers. In order to identify potential strains with good antimicrobial activity and to advance the development of microalgae as antimicrobial agents, a rigorous scientific approach is required. Microalgae are most commonly screened for antimicrobial activity using the disc diffusion assay but this assay is problematic and produces false-positive and false-negative results. Quantitative minimum inhibitory concentration (MIC) values generated in assays such as the microdilution broth assay are more reproducible and enable comparison of results between research groups. For the present review, a dataset was compiled of published MIC values for microalgae. The Cyanobacteria and Chlorophyta were the best represented and other phyla were under represented. This data was used for assessment of factors influencing antimicrobial activity, including test microorganisms, microalgae taxonomy, different solvents for extraction and the growth phase at harvest. Activity was considered good if MIC values were < 1 mg/mL, moderate if MIC values were 1-8 mg/mL and weak with MIC >8.0 mg/mL. Areas requiring more research are discussed including screening a greater diversity of species in appropriate assays, reporting negative results, testing the culture supernatant for activity, synergistic effects and identifying antimicrobial compounds in the Chlorophyta. The potential for successful development and commercialization of microalgae antimicrobial agents will increase as more microalgae are screened and compounds identified.

Effect of storage on plant biostimulant and bioactive properties of freeze-dried Chlorella vulgaris biomass.

Microalgae are potential plant biostimulants and biocontrol agents. A major hurdle towards their commercialization is the production of large volumes of biomass at the correct time of year. Secondary metabolites are unstable and the "shelf-life" of bioactive microalgal biomass needs to be investigated. The aim of the study was to investigate the effects of storage conditions on freeze-dried microalgae to determine how long the biomass retained its growth promoting and bioactive properties under various temperature and light conditions. Chlorella vulgaris biomass was stored in the dark at - 70 °C, 10 °C, and 25 °C and in the light at 25 °C. Samples were tested every 3-4 months for 15 months. Storage time significantly influenced the rate of change in the bioactivity in the C. vulgaris biomass with storage temperature also having some effect. Rooting activity decreased in the mungbean rooting assay over time up to 12 months and then increased slightly. Antimicrobial activity increased against Staphylococcus aureus and Escherichia coli for up to 12 months and then declined. Antioxidant activity measured in the DPPH assay remained relatively stable for up to 12 months and then significantly decreased with longer storage. The change in bioactivity over time was attributed to the gradual breakdown of the rigid cell wall of C. vulgaris, thereby improving extraction efficiency but exposing the secondary metabolites to oxygen, thus quickening their degradation. Biomass produced for commercial purposes requires preliminary validation as the results of the present study showed that bioactive compounds are susceptible to degradation over time. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10811-021-02596-9.

Interactive effects of plant growth-promoting rhizobacteria and a seaweed extract on the growth and physiology of Allium cepa L. (onion).

Detrimental effects caused by the overuse of synthetic agrochemicals have led to the development of natural biostimulants such as seaweed extracts and plant growth-promoting rhizobacteria (PGPR) being used as an alternative, environmentally-friendly technology to improve crop growth and increase agricultural yields. The present study aimed to investigate the interactions between PGPR and a commercial seaweed extract on the growth and biochemical composition of onion (Allium cepa). A pot trial was conducted under greenhouse conditions where onion plants were treated individually with the two PGPR, namely Bacillus licheniformis (BL) and Pseudomonas fluorescens (PF) and a seaweed extract Kelpak® (KEL) and combinations of KEL + BL and KEL + PF. Growth and yield parameters were measured after 12 weeks. KEL-treated plants showed the best growth response and overcame the inhibitory effects of BL treatment. KEL-treated plants also had the highest chlorophyll content. PGPR application improved the mineral nutrition of onion with these plants having the highest mineral content in the leaves and bulb. All biostimulant treatments increased the endogenous cytokinin and auxin content with the highest concentrations generally detected in the PF-treated plants. These results suggest that co-application of different biostimulant classes with different modes of action could further increase crop productivity with an improvement in both growth and nutrition content being achieved in onion with the co-application of a seaweed extract and PGPR.

Potential of phytohormones as a strategy to improve microalgae productivity for biotechnological applications.

Microalgae have many commercial applications including biofuel production, use in human and animal nutrition, as pharmaceuticals and therapeutic compounds, in beauty-related products and as biofertilizers in the agricultural sector. However, more research needs to be directed to reducing production costs in an environmentally friendly way before their commercial potential can be fully realized. This review discusses the current knowledge on the occurrence and physiological roles of phytohormones in microalgae and explores the potential of phytohormone treatments to improve cultivation practices, including increasing lipid content and productivity for biofuel production. Microalgae synthesize a wide array of phytohormones and are able to regulate the levels of active phytohormones. Biosynthetic and conversion pathways share some key components but are more rudimentary compared to vascular plants and are likely to have some unidentified conjugation mechanisms. Phytohormones have a dual function in microalgae. Specific phytohormones are involved in the regulation of the cell cycle and other metabolic processes, influencing biomass and primary metabolite accumulation. They are also involved in responses to abiotic stresses, allowing microalgae to adapt to the prevailing conditions. Phytohormones provide a promising strategy to improve mass culture biotechnology due to their intrinsic role in microalgal growth and survival, increasing lipid productivity and improving their tolerance to more extreme environmental changes. This makes them less susceptible to environmental fluctuations. This improves the productivity of the cultures for biofuel production and would be beneficial in a biorefinery approach. Overall, phytohormones provide an exciting and promising avenue of research to improve microalgae cultivation, taking it a step closer to successful commercialization.

Effect of cell disruption methods on the extraction of bioactive metabolites from microalgal biomass.

Microalgae synthesize a variety of potentially high-value compounds. Due to their robust cell wall, cell disruption is necessary to improve extraction of these compounds. While cell disruption methods have been optimized for lipid and protein extraction, there are limited studies for other bioactive compounds. The present study investigated the effect of freeze-drying combined with sonication or ball-milling on the extraction of antioxidant and plant biostimulating compounds from Chlorella sp., Chlorella vulgaris and Scenedesmus acutus. Both cell disruption methods resulted in higher extract yields from the biomass compared to freeze-dried biomass using 50% methanol as a solvent. Antioxidant activity of Chlorella extracts was generally higher than freeze-dried extracts based on the diphenylpicrylhydrazyl (DPPH) and ß-carotene linoleic acid assays. However, the effectiveness of each treatment varied between microalgae strains. Sonication resulted in the highest antioxidant activity in Chlorella sp. extracts. Ball-milling gave the best results for C. vulgaris extracts in the DPPH assay. Both cell disruption methods decreased antioxidant activity in S. acutus extracts. Plant biostimulating activity was tested using the mung bean rooting assay. Damaging the membrane by freeze-drying was sufficient to release the active compounds using water extracts. In contrast, both cell disruption methods negatively affected the biological activity of the extracts. These results indicate that bioactive compounds in microalgae are sensitive to post-harvest processes and their biological activity can be negatively affected by cell disruption methods. Care must be taken to not only optimize yield but to also preserve the biological activity of the target compounds.

Can the use of natural biostimulants be a potential means of phytoremediating contaminated soils from goldmines in South Africa?

Biostimulants offer great potential in improving phytoremediation of contaminated soils. In the current greenhouse-based study, Brassica juncea seedlings grown on soils collected from Krugersdorp Goldmine and the adjourning areas (a Game Reserve and private farmland) were supplemented with different biostimulants (Kelpak® = KEL, vermicompost leachate = VCL, smoke-water = SW). Indole-3-butyric acid (IBA) was included in the study for comparative purposes because these biostimulants are known to enhance rooting. Prior to the pot trial, concentrations of elements in the three soil types were determined using Inductively Coupled Plasma-Optical Emission Spectroscopy. Plants were harvested after 105 days and the growth and concentrations of elements in the various plant organs were determined. TheB. juncea seedlings with and without biostimulants did not survive when growing in soil from the Krugersdorp Goldmine. The Game Reserve and private farmland soils supplemented with KEL produced the highest plant biomass and the lowest accumulation of metals in the organs of B. juncea. High concentrations (>13 000 mg kg(-1)) of zinc and aluminium were quantified in the roots of IBA-supplemented soils from the Game Reserve. Generally, IBA and SW enhanced the phytoremediation of B. juncea due to elevated levels of elements that accumulated in their different organs.

Cytokinin profiles in ex vitro acclimatized Eucomis autumnalis plants pre-treated with smoke-derived karrikinolide.

KEY MESSAGE: The current evidence of regulatory effect of smoke-water (SW) and karrikinolide (KAR(1)) on the concentrations of endogenous cytokinins in plants partly explain the basis for their growth stimulatory activity. Karrikinolide (KAR1) which is derived from smoke-water (SW) is involved in some physiological aspects in the life-cycle of plants. This suggests a potential influence on the endogenous pool (quantity and quality) of phytohormones such as cytokinins (CKs). In the current study, the effect of SW (1:500; 1:1000; 1:1500 v/v dilutions) and KAR1 (10(-7); 10(-8); 10(-9) M) applied during micropropagation of Eucomis autumnalis subspecies autumnalis on the ex vitro growth and CKs after 4 months post-flask duration was evaluated. The interactions of SW and KAR(1) with benzyladenine (BA), α-naphthaleneacetic acid (NAA) or BA+NAA were also assessed. Plants treated with SW (1:500) and KAR1 (10(-8) M) demonstrated superior growth in terms of the rooting, leaf and bulb sizes and fresh biomass than the control and plants treated with BA and BA+NAA. However, plant growth was generally inhibited with either SW (1:500) or KAR1 (10(-8) M) and BA when compared to BA (alone) treatment. Relative to NAA treatment, the presence of KAR(1) (10(-7) M) with NAA significantly increased the leaf area and fresh biomass. Both SW and KAR1-treated plants accumulated more total CKs, mainly isoprenoid-type than the control and NAA-treated plants. The highest CK content was also accumulated in SW (1:500) with BA+NAA treatments. Similar stimulatory effects were observed with increasing concentrations of KAR(1) and BA. The current findings establish that SW and KAR1 exert significant influence on the endogenous CK pools. However, the better growth of plants treated with SW and KAR1 treatments was not exclusively related to the endogenous CKs.

Effects of Cd and Al stress on secondary metabolites, antioxidant and antibacterial activity of Hypoxis hemerocallidea Fisch. & C.A. Mey.

This study investigated the effects of cadmium (Cd) and aluminium (Al) on the accumulation of phenolics, flavonoids and the bioactive compound hypoxoside in Hypoxis hemerocallidea. In addition, antioxidant scavenging and antibacterial activity were assessed to evaluate if Cd and Al stress affect the accumulation of bioactive compounds in H. hemerocallidea. In vitro grown plantlets of H. hemerocallidea were acclimatized for seven months in a greenhouse. Thereafter plants were exposed to various concentrations of Cd and Al both singularly and in combination in the form of Cd(NO3)2 (2, 5, 10 mg Cd/L); Al3(NO3)3 (500, 1000, 1500 mg Al/L) and combinations of Cd and Al (Cd 2:Al 500, Cd 5:Al 1000 and Cd 10:Al 1500 mg/L) for a further six weeks. The highest amounts of Cd and Al translocated to the shoot were 34 and 1608 mg/L respectively. Phytochemical screening showed significantly high amounts of total phenolics and flavonoids at the moderate Cd treatment (5 mg/L) compared to the controls. Exposure to Cd and Al significantly decreased the accumulation of hypoxoside. There was a significant increase in diphenylpicrylhydrazyl (DPPH) antioxidant scavenging activity in most of the metal-treated plants compared to the positive control ascorbic acid. Extracts from Cd 2 mg/L treatment exhibited moderate antibacterial activity against Staphylococcus aureus compared to the control. The results of the present study revealed that cultivating H. hemerocallidea on metal contaminated soils affects the accumulation of the bioactive compound, hypoxoside.

Dissecting the role of two cytokinin analogues (INCYDE and PI-55) on in vitro organogenesis, phytohormone accumulation, phytochemical content and antioxidant activity.

There is a continuous search for new chemical entities to expand the collection of suitable compounds to increase the efficiency of micropropagation protocols. Two cytokinin (CK) analogues, 2-chloro-6-(3-methoxyphenyl)aminopurine (INCYDE) and CK antagonist 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) were used as a tool to elucidate the auxin-CK crosstalk under in vitro conditions in the medicinally important plant, Eucomis autumnalis subspecies autumnalis. These compounds were tested at 0.01, 0.1 and 10 µM alone as well as in combination with benzyladenine (BA) and naphthaleneacetic acid (NAA). The organogenesis, phytohormone content, phytochemical and antioxidant response in 10 week-old-in vitro regenerated E. autumnalis subspecies autumnalis was evaluated. INCYDE generally favoured shoot regeneration while the effect of PI-55 was more evident in root proliferation. Overall, INCYDE promoted the accumulation of higher concentrations and varieties of endogenous CK relative to the PI-55 treatments. In contrast, higher concentration of indole-3-acetic acid and 2-oxindole-3-acetic acid were generally observed in PI-55-supplemented cultures when compared to plantlets derived from INCYDE. Both CK analogues (individually and in-conjunction with exogenously applied PGRs) significantly influenced the phytochemicals and consequently the antioxidant potential of the in vitro regenerants. These results provided insight on how to alleviate root inhibition, a problem which causes considerable loss of several elite species during micropropagation.

Manipulation of nitrogen levels and mode of cultivation are viable methods to improve the lipid, fatty acids, phytochemical content, and bioactivities in Chlorella minutissima.

The effect of nitrogen (N: low = 2% N or moderate = 10% N) levels and cultivation (photoautotrophic or mixotrophic) modes on the biochemicals in Chlorella minutissima was evaluated using a mass culture system. Moderate N and mixotrophic cultures had higher biomass, protein, carbohydrate content and photosynthetic pigments than the low N and photoautotrophic treatments. In contrast, lipid and fatty acid content of the low N and photoautotrophic treatments were higher than in the moderate N and mixotrophic cultures. More phytochemicals were accumulated in moderate N and mixotrophic cultures which corresponded to better antioxidant capacity in the extracts. The most potent (0.7 mg · mL(-1) ) acetylcholinesterase inhibitory activity was displayed by moderate N and mixotrophic treatment. Approximately 60% of the extracts exhibited a noteworthy antimicrobial activity regardless of the N levels and cultivation modes. Thus, moderate N level enhanced the phytochemicals and biological activities of C. minutissima cultured under a mixotrophic system.

Advances in algal drug research with emphasis on enzyme inhibitors.

Enzyme inhibitors are now included in all kinds of drugs essential to treat most of the human diseases including communicable, metabolic, cardiovascular, neurological diseases and cancer. Numerous marine algae have been reported to be a potential source of novel enzyme inhibitors with various pharmaceutical values. Thus, the purpose of this review is to brief the enzyme inhibitors from marine algae of therapeutic potential to treat common diseases. As per our knowledge this is the first review for the potential enzyme inhibitors from marine origin. This review contains 86 algal enzyme inhibitors reported during 1989-2013 and commercial enzyme inhibitors available in the market. Compounds in the review are grouped according to the disease conditions in which they are involved; diabetes, obesity, dementia, inflammation, melanogenesis, AIDS, hypertension and other viral diseases. The structure-activity relationship of most of the compounds are also discussed. In addition, the drug likeness properties of algal inhibitors were evaluated using Lipinski's 'Rule of Five'.

Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss.

Alpha-glucosidase inhibitors play a potential role in the treatment of type 2 diabetes by delaying glucose absorption in the small intestine. Ecklonia maxima, a brown alga which grows abundantly on the west coast of South Africa, is used to produce alginate, animal feed, nutritional supplements and fertilizer. The crude aqueous methanol extract, four solvent fractions and three phlorotannins: 1,3,5-trihydroxybenezene (phloroglucinol) (1), dibenzo [1,4] dioxine-2,4,7,9-tetraol (2) and hexahydroxyphenoxydibenzo [1,4] dioxine (eckol) (3) isolated from E. maxima were evaluated for antiradical and alpha-glucosidase inhibitory activities. All the phlorotannins tested had strong antioxidant activities on DPPH free radicals with EC50 values ranging from 0.008 to 0.128µM. Compounds 2 and 3 demonstrated stronger antioxidant activity and an alpha-glucosidase inhibitory property than positive controls. These results suggest that E. maxima could be a natural source of potent antioxidants and alpha-glucosidase inhibitors. This study could facilitate effective utilization of E. maxima as an oral antidiabetic drug or functional food ingredient with a promising role in the formulation of medicines and nutrition supplements.

Plant growth regulators enhance gold uptake in Brassica juncea.

The use of plant growth regulators is well established and they are used in many fields of plant science for enhancing growth. Brassica juncea plants were treated with 2.5, 5.0 and 7.5 microM auxin indole-3-butyric acid (IBA), which promotes rooting. The IBA-treated plants were also sprayed with 100 microM gibberellic acid (GA3) and kinetin (Kin) to increase leaf-foliage. Gold (I) chloride (AuCl) was added to the growth medium of plants to achieve required gold concentration. The solubilizing agent ammonium thiocyanate (1 g kg(-1)) (commonly used in mining industries to solubilize gold) was added to the nutrient solution after six weeks of growth and, two weeks later, plants were harvested. Plant growth regulators improved shoot and root dry biomass of B. juncea plants. Inductively Coupled Plasma Optical Emission Spectrometry analysis showed the highest Au uptake for plants treated with 5.0 microM IBA. The average recovery of Au with this treatment was significantly greater than the control treatment by 45.8 mg kg(-1) (155.7%). The other IBA concentrations (2.5 and 7.5 microM) also showed a significant increase in Au uptake compared to the control plants by 14.7 mg kg(-1) (50%) and 42.5 mg kg(-1) (144.5%) respectively. A similar trend of Au accumulation was recorded in the roots of B. juncea plants. This study conducted in solution culture suggests that plant growth regulators can play a significant role in improving phytoextraction of Au.

Auxin and cytokinin relationships in 24 microalgal strains(1).

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection-41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC-712 the lowest biomass. The auxins, indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49 nmol IAA · g(-1) DW and 0.18 to 99.83 nmol IAM · g(-1) DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29 nmol · g(-1) DW in Klebsormidium flaccidum MACC-692 to 21.40 nmol · g(-1) DW in Stigeoclonium nanum MACC-790. The general trend was that cis-zeatin types were the predominant cytokinins; isopentenyladenine-type cytokinins were present in moderate concentrations, while low levels of trans-zeatin-type and very low levels of dihydrozeatin-type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O-glucosides were low. Only one N-glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2- to 4-fold higher than the cytokinin content.

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes.

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N6-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N6-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.

Cadmium induces hypodermal periderm formation in the roots of the monocotyledonous medicinal plant Merwilla plumbea.

BACKGROUND AND AIMS: Merwilla plumbea is an important African medicinal plant. As the plants grow in soils contaminated with metals from mining activities, the danger of human intoxication exists. An experiment with plants exposed to cadmium (Cd) was performed to investigate the response of M. plumbea to this heavy metal, its uptake and translocation to plant organs and reaction of root tissues. METHODS: Plants grown from seeds were cultivated in controlled conditions. Hydroponic cultivation is not suitable for this species as roots do not tolerate aquatic conditions, and additional stress by Cd treatment results in total root growth inhibition and death. After cultivation in perlite the plants exposed to 1 and 5 mg Cd L(-1) in half-strength Hoagland's solution were compared with control plants. Growth parameters were evaluated, Cd content was determined by inductively coupled plasma mass spectroscopy (ICP-MS) and root structure was investigated using various staining procedures, including the fluorescent stain Fluorol yellow 088 to detect suberin deposition in cell walls. KEY RESULTS: The plants exposed to Cd were significantly reduced in growth. Most of the Cd taken up by plants after 4 weeks cultivation was retained in roots, and only a small amount was translocated to bulbs and leaves. In reaction to higher Cd concentrations, roots developed a hypodermal periderm close to the root tip. Cells produced by cork cambium impregnate their cell walls by suberin. CONCLUSIONS: It is suggested that the hypodermal periderm is developed in young root parts in reaction to Cd toxicity to protect the root from radial uptake of Cd ions. Secondary meristems are usually not present in monocotyledonous species. Another interpretation explaining formation of protective suberized layers as a result of periclinal divisions of the hypodermis is discussed. This process may represent an as yet unknown defence reaction of roots when exposed to elemental stress.

CHANGES IN ENDOGENOUS CYTOKININ CONCENTRATIONS IN CHLORELLA (CHLOROPHYCEAE) IN RELATION TO LIGHT AND THE CELL CYCLE(1).

Endogenous cytokinins were quantified in synchronized Chlorella minutissima Fott et Novákova (MACC 361) and Chlorella sp. (MACC 458) grown in a 14:10 light:dark (L:D) photoperiod. In 24 h experiments, cell division occurred during the dark period, and cells increased in size during the light period. Cytokinin profiles were similar in both strains, consisting of five cis-zeatin (cZ) and three N(6) -(2-isopentenyl)adenine (iP) derivatives. Cytokinin concentrations were low during the dark period and increased during the light period. In 48 h experiments using synchronized C. minutissima (MACC 361), half the cultures were maintained in continuous dark conditions for the second photoperiod. Cell division occurred during both dark periods, and cells increased in size during the light periods. Cultures kept in continuous dark did not increase in size following cell division. DNA analysis confirmed these results, with cultures grown in light having increased DNA concentrations prior to cell division, while cultures maintained in continuous dark had less DNA. Cytokinins (cZ and iP derivatives) were detected in all samples with concentrations increasing over the first 24 h. This increase was followed by a large increase, especially during the second light period where cytokinin concentrations increased 4-fold. Cytokinin concentrations did not increase in cultures maintained in continuous dark conditions. In vivo deuterium-labeling technology was used to measure cytokinin biosynthetic rates during the dark and light periods in C. minutissima with highest biosynthetic rates measured during the light period. These results show that there is a relationship between light, cell division, and cytokinins.