High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N(6)-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).

Evolutionary origin of highly repetitive plastid genomes within the clover genus (Trifolium).

BACKGROUND: Some clover species, particularly Trifolium subterraneum, have previously been reported to have highly unusual plastomes, relative to closely related legumes, enlarged with many duplications, gene losses and the presence of DNA unique to Trifolium, which may represent horizontal transfer. In order to pinpoint the evolutionary origin of this phenomenon within the genus Trifolium, we sequenced and assembled the plastomes of eight additional Trifolium species widely sampled from across the genus. RESULTS: The Trifolium plastomes fell into two groups: those of Trifolium boissieri, T. strictum and T. glanduliferum (representing subgenus Chronosemium and subg. Trifolium section Paramesus) were tractable, assembled readily and were not unusual in the general context of Fabeae plastomes. The other Trifolium species ("core Trifolium") proved refractory to assembly mainly because of numerous short duplications. These species form a single clade, which we call the "refractory clade" (comprising subg, Trifolium sections Lupinaster, Trifolium, Trichocephalum, Vesicastrum and Trifoliastrum). The characteristics of the refractory clade are the presence of numerous short duplications and 7-15% longer genomes than the tractable species. Molecular dating estimates that the origin of the most recent common ancestor (MRCA) of the refractory clade is approximately 13.1 million years ago (MYA). This is considerably younger than the estimated MRCA ages of Trifolium (c. 18.6 MYA) and Trifolium subg. Trifolium (16.1 MYA). CONCLUSIONS: We conclude that the unusual repetitive plastome type previously characterized in Trifolium subterraneum had a single origin within Trifolium and is characteristic of most (but not all) species of subgenus Trifolium. It appears that an ancestral plastome within Trifolium underwent an evolutionary change resulting in plastomes that either actively promoted, were permissive to, or were unable to control, duplications within the genome. The precise mechanism of this important change in the mode and tempo of plastome evolution deserves further investigation.

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements.

Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes.

BACKGROUND AND AIMS: Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. METHODS: Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. KEY RESULTS: Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. CONCLUSIONS: Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.

New synthetic pyridine derivate as potential elicitor in production of isoflavonoids and flavonoids in Trifolium pratense L. suspension culture.

The production of secondary metabolites in Trifolium pratense L. suspension culture of the family of legume plants (Fabaceae) is low, and therefore there was an attempt to increase it by elicitation. New synthetic substance, 2-(2-fluoro-6-nitrobenzylsulfanyl)pyridine-4-carbothioamide, was tested as elicitor--a substance that showed the best elicitation effect after 48-hour application of 1 µmol L⁻¹ concentration. Maximum contents of genistin (11.60 mg g⁻¹ DW), daidzein (8.31 mg g⁻¹ DW), and genistein (1.50 mg g⁻¹ DW) were recorded, and the production of these isoflavonoids thus significantly increased, when compared with the control, by 152%, 151%, and 400%. The maximum content of flavonoids (5.78 mg g⁻¹ DW) and the increase in the production by 142%, when compared with the control, were induced by 6-hour application of 100 µmol L⁻¹ concentration. The tested substance showed to be an effective elicitor of phenylpropane metabolism.

Using the pollen viability and morphology for fluoride pollution biomonitoring.

The methods using plants for biomonitoring of air and soil quality are simple, cheap, and fast and can supplement the classical physicochemical methods. In this study, biological pollen characterization of some collected legume species from an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual value of pollen as a bioindicator of the effects of soil and atmospheric pollution. Young buds and flowers of six legumes (Cercis siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants were removed and compared. Studies of light and electron microscopic preparation showed some abnormalities during pollen development in affect of fluoride pollution. The viability of pollen grains estimated by staining with acetocarmine shows sharp differences in smearing advanced pollen grains from abnormal ones. Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R. pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light, partial, or no staining with acetocarmine, whereas almost all of the control ones clearly stained. Observation of the pollen grains by light microscopy and scanning electron microscopy showed the significant effect of fluoride on shapes and sizes of pollen grains. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration. Therefore, pollen grains provide essential information on biological impact of pollutants and they are good candidates for biomonitoring the atmospheric and edaphic pollutions.

Resistance of red clover (Trifolium pratense) to the root parasitic plant Orobanche minor is activated by salicylate but not by jasmonate.

BACKGROUND AND AIMS: Obligate root holoparasites of the genus Orobanche attack dicotyledonous crops and cause severe losses in many parts of the world. Chemical induction of plant defence systems such as systemic acquired resistance was proposed to be an available strategy to control the root parasite, but the detailed mechanisms involved have not been clarified. The aim of this study was to elucidate the effects of salicylic acid (SA), jasmonic acid (JA) and their analogues on resistance of red clover to Orobanche parasitism. METHODS: Roots of red clover grown in plastic chambers were applied with SA, S-methyl benzo[1,2,3]thiadiazole-7-carbothioate (BTH), methyl jasmonate (MeJA) and n-propyl dihydrojasmonate (PDJ), and then were inoculated with O. minor seeds. Attachments of the parasite were observed after 5 weeks. KEY RESULTS: SA and BTH, inducers of SA-mediated defences, significantly reduced the number of established parasites by more than 75 %. By contrast, MeJA and PDJ, inducers of JA-mediated defences, did not affect parasitism. The reduction in the number of established parasites by SA and BTH was due to the inhibited elongation of O. minor radicles and the activation of defence responses in the host root including lignification of the endodermis. CONCLUSIONS: These results suggest that SA-induced resistance, but not JA-induced resistance, is effective in inhibiting Orobanche parasitism and that the resistance is expressed by the host root both externally and internally.

Red clover (Trifolium pratense).

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with a high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express a beta-glucuronidase (GUS) reporter gene and to silence an endogenous polyphenol oxidase gene in red clover.

Molecular cloning, functional expression in Escherichia coli and enzymatic characterisation of a cysteine protease from white clover (Trifolium repens).

This paper presents the cloning and biochemical characterisation of the cysteine protease Tr-cp 14 from white clover (Trifolium repens). The predicted amino acid sequence of Tr-cp 14 is 71%, 74% and 74% identical to the cysteine proteases XCP1 and XCP2 from Arabidopsis thaliana, and p48h-17 from Zinnia elegans, respectively. These cysteine proteases have previously been shown to be involved in programmed cell death during tracheary element differentiation. The precursor polypeptide of Tr-cp 14 was expressed in Escherichia coli, purified from inclusion bodies and refolded. The precursor polypeptide could be processed to its active mature form autocatalytically at pH 5.0 and had a requirement for 20 mM l-cysteine for optimal activity. Mature Tr-cp 14 showed a preference for synthetic aminomethylcoumarin substrates with either Leu or Phe in the P2 position when tested with Arg in P1. A substrate with Arg in both the P1 and P2 position was not accepted as substrate.

Effects of a cold treatment of the root system on white clover (Trifolium repens L.) morphogenesis and nitrogen reserve accumulation.

The ability of white clover (Trifolium repens L.) to undergo cold acclimation is an important determinant of its persistence in mixed swards since growth rate at low temperatures sustains higher clover contents at the start of spring. During a re-growth period following defoliation, a gradual exposure of the root system (cv. Grasslands Huia) led to some physiological and morphological changes of cold-adaptive significance, similar to those developed by clover ecotypes originating in northern areas of Europe. Thus, cold exposure of the root system resulted in small-leaved prostrate forms of white clover after one month of re-growth. Similarly, cold exposure increased the ability of plants to store nitrogen since the application of low temperatures to the root system enhanced soluble protein accumulation in roots and in stolons. More specifically, cold exposure of the roots induced gene expression of a vegetative storage protein (17.3 kDa VSP) in both organs. These results demonstrate that the root system of clover plants should be a site of perception of the low-temperature stimulus, and gave rise to the question of the transduction of the cold signal from the roots to the aerial parts. On the basis of this study and taking into account molecular aspects concerning the clover VSP, it is suggested that this protein could participate in cold acclimation in addition to its role in nitrogen storage.

Modular construction of the protoderm and peripheral root cap in the "open" root apical meristem of Trifolium repens cv. Ladino.

Roots with open apical organization are defined by not having specific tiers of initial cells in the root apical meristem; those with closed apical organization have specific initial tiers to which all cell files can be traced. An example of the clear organization of closed roots is the development protocol of the root cap and protoderm. The key event in differentiating these tissues is the T-division, a periclinal division of the root cap/protoderm (RCP) initial that establishes a module. Each module comprises two packets, the protoderm and peripheral root cap. Consecutive T-divisions of the same RCP initial produce up to five modules on average in a lineage of cells in white clover (Trifolium repens cv. Ladino), with all lineages around the circumference of the root dividing in "waves" to form one module prior to the next. On average, clover has approximately 32 axial protoderm and peripheral root cap cells in each module, and 32 RCP lineages. The occurrence of RCP T-divisions in white clover, a root with open apical organization, and the subsequent modular construction of the root cap and protoderm, provides a link between open and closed roots and suggests a common developmental feature that most roots of seed plants may share independent of their root meristem organization type. The open apical organization of the white clover root varies from roots with closed apical organization in that the RCP initials occur in staggered positions instead of connected to discrete tiers, and the peripheral root cap and columella daughter cells form additional layers of cells. White clover also forms root hairs on all protoderm cells irrespective of their position relative to the underlying cortical cells.