Hector E. Flores

‘Radicle' biochemistry: the biology of root-specific metabolism

The roots of higher plants are a fascinating and largely unexplored biological frontier. One of their features is the ability to synthesize a remarkable diversity of secondary metabolites, and to adjust their metabolic activities in response to biotic and abiotic stress. This includes the ability to exude a complex array of micro- and macromolecules into the rhizosphere, with the potential to affect the inter-relationships between plants and beneficial or deleterious soil-borne organisms. In the past, research on root biology has been hampered by the underground growth habit of roots and by the lack of a suitable experimental system. However, recent progess in growing roots in isolation has greatly facilitated the study of root-specific metabolism and contributed to our understanding of this remarkable plant organ.

Root-specific metabolism: The biology and biochemistry of underground organs

SummaryThe roots of higher plants comprise a metabolically active and largely unexplored biological frontier. Some of their prime features include the ability to synthesize a remarkably diverse group of secondary metabolites, and to adjust their metabolic activities in response to different abiotic and biotic stresses. This adjustment includes the ability to exude a wide array of micro- and macromolecules into the rhizosphere and to phytoremediate toxic metals, with the potential to affect and alter the relationships between plants and both beneficial and deleterious soil-borne pathogens. In the past, research on root biology has been hampered by the underground nature of roots and the lack of suitable experimental systems to study root-root and root-microbe communications. However, recent progress in growing roots in isolation with other elements of the rhizosphere has greatly facilitated the study of root-specific metabolism and contributed to our understanding of this organ.

Ocatin. A Novel Tuber Storage Protein from the Andean Tuber Crop Oca with Antibacterial and Antifungal Activities

The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, andPseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum,Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens.

Green Roots: Photosynthesis and Photoautotrophy in an Underground Plant Organ.

The potential for photosynthetic and photoautotrophic growth was studied in hairy root cultures of Asteraceae and Solanaceae species. Upon transfer to light, initially heterotrophic root cultures of Acmella oppositifolia and Datura innoxia greened rapidly, differentiated chloroplasts, and developed light-dependent CO2 fixation in the cortical cells. Photosynthetic potential was expressed in root cultures of all the Asteraceae genera examined (Acmella, Artemisia, Rudbeckia, Stevia, and Tagetes). Hairy roots of A. oppositifolia and D. innoxia were further adapted to photoautotrophy by growing in the presence of light and added CO2 (1–)5%) and by direct or sequential transfers into media containing progressively lower sugar concentrations. The transition to photoautotrophy was accompanied by an increase in CO2 fixation and in the specific activity of 1,5-ribulose-bisphosphate carboxylase/ oxygenase (Rubisco). During the adaptation of A. oppositifolia roots to photoautotrophy, the ratio of Rubisco to phosphoenolpyruvate carboxylase increased significantly, approaching that found in the leaves. The levels and patterns of alkaloids and polyacetylenes produced by Solanaceae and Asteraceae hairy roots, respectively, were dramatically altered in photomixotrophic and photoautotrophic cultures. Photoautotrophic roots of A. oppositifolia have been mainitained in vitro for over 2 years.

Primary and Secondary Metabolism of Polyamines in Plants

The diamine putrescine and the polyamines spermidine and spermine are amino acid-derived, aliphatic nitrogenous compounds of wide distribution among plant cells (Fig.1). The earliest reference to polyamines in the scientific literature is van Leeuwenhoek’s classic letter to the Royal Society of London in 1678. During his studies describing spermatozoa, he observed the gradual formation of colorless crystals upon drying the samples. The correct structure of these crystals, which corresponded to spermine, was not determined until over 250 years later.1 Spermidine was later found in mammalian tissues. The related diamines putrescine and cadaverine were found in decomposing animal and vegetable matter as a result of microbial activity. Within this historical frame, it is not surprising that earlier studies on these compounds were done mostly on non-plant systems. Recent interest in the function of polyamines in higher plants is in good part derived from discoveries in microbial and animal cells.

Biosynthesis of Defense-Related Proteins in Transformed Root Cultures of Trichosanthes kirilowii Maxim. var japonicum (Kitam.)

We have established transformed (“hairy”) root cultures from Trichosanthes kirilowii Maxim. var japonicum Kitam. (Cucurbitaceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other root-specific defense-related plant proteins. Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacterium rhizogenes American Type Culture Collection strain 15834. Each species accumulated reproducibly a discrete protein pattern in the culture medium. Analysis of the extracellular proteins from T. kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures. Maximum protein accumulation, approaching 20 [mu]g/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase. Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class III chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class III chitinases. The Trichosanthes chitinases also showed reactivity with a cucumber class III chitinase antiserum and chitinolytic activity in a glycol chitin gel assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed T. kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein). Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein. Roots undergoing secondary growth and differentiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.

In vitro culture and precocious germination ofTaxus embryos

SummaryThe lengthy dormancy requirement of yew seeds can be overcome with a simple in vitro method. Viable embryos were excised from seeds ofTaxus brevifolia and four cultivars ofT. media over a range of developmental stages. Embryos were cultured in several basal media formulations (Whites’, Gamborg’s B5 and Murashige and Skoog’s) under dark or light. After a lag period of 1 to 2 wk, embryos of both species germinated precociously. Germination rates of up to 70% were obtained withT. media cv. Hicksi embryos. The highest rates of germination were obtained in White’s and MS media. Embryos excised from green seeds with undeveloped arils showed the highest germination rates. As the seeds approached maturity, in vitro germination rates of the excised embryos declined dramatically. Green seeds and seeds with developing arils could be stored at 5° C without large loss in embryo germination. Seeds with fully developed arils could be stored frozen at −20° C for 1 wk while still allowing about 50% of embryo germination. At least 30% of the precociously germinated embryos of both species were able to develop into full seedlings. Our method appears to be generally applicable toTaxus spp.

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473–497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.

Stimulation of ethylene production by catecholamines and phenylethylamine in potato cell suspension cultures

The catecholamines (50 μM dopamine, 50 μM norepinephrine and 100 μM epinephrine) and phenylethylamine (200 μM) were found to stimulate ethylene production in potato suspension cultures. When 100 μM amino-oxyacetic acid was added together with epinephrine, ethylene release returned to control levels. The endogenous 1-aminocyclopropane-1-carboxylic acid levels were increased in parallel with the release of ethylene, suggesting that the observed effect probably occurs via regulation of aCC synthase. Our results suggest that there is a link between these naturally occurring monoamines and ethylene in plants.

Embryo culture and taxane production inTaxus spp

SummaryWe have previously shown (Flores and Sgrignoli, 1991) that immature embryos ofTaxus brevifolia andT. X media are capable of precocious germination and can grow into seedlings in vitro. The cultural and environmental parameters for embryo germination and conversion into seedlings have been optimized and extended toT. baccata andT. cuspidata. A 14-h photoperiod improved embryo germination and growth into seedlings. A pregermination cold treatment of the seeds had a positive effect on both the onset and percentage of germination. Embryos from cold-treated seeds germinated earlier and at a higher frequency than those from control seeds. Boron was necessary for embryo germination, and levels of this micronutrient were established for optimal growth and germination ofT. brevifolia andT. X media cv. Hicksii embryos. Gupta and Durzan’s medium was superior to White’s for embryo germination and root formation. Naphthaleneacetic acid stimulated root formation in embryo-derived seedlings. We also found that immature embryos could be induced to form callus with embryogenic potential. Taxol and related taxanes were detected in embryo- derived seedlings.