Hormonal effects on phyllotaxis ofEuphorbia lathyris L.

Benzyladenine and gibberellic acid sprays (100 mg/l) onEuphorbia lathyris L. plants were absolutely successful (100% of the plants) in inducing a change in its normal decussate phyllotaxis. Benzyladenine produced a change to tetracussate, tricussate and bijugate, and gibberellic acid to spiral phyllotaxis. Benzyladenine and gibberellic acid treatments resulted in a significant increase in apex diameter (72.8% and 19.1% respectively). CCC, Ancymidol, Alar and Glyphosine did not alter decussate phyllotaxis.     

Jasmonic Acid induces tuberization of potato stolons cultured in vitro

The aim of the study was to assess the potential in vitro effects of jasmonic acid and kinetin on tuberization of potato (Solanum tuberosum). Of the two, the former was by far the stronger in vitro promoter of stolon tuberization. Number of tubers induced per stolon, tuberization rate, and final tuber weight were higher by factors of 2.8, 2.3, and 6.4, respectively. Bioassay sensitivity of jasmonic acid, measured in terms of the point at which the concentration for inducing tuberization was saturating, was more than 20 times greater than that of kinetin. Tuberization in both cases was associated with a decrease in rooting ability. Jasmonic acid also triggered a general state of induction throughout the stolon.     

The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts

We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0.3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-α (TNF-α) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant.     

Effect of carbon dioxide and ethylene on tuberization of isolated potato stolons cultured in vitro

Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C₂H₄ which is by itself also inhibitory of tuberization. Furthermore, C₂H₄ inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C₂H₄. The antagonistic actions of CO₂ and C₂H₄ on tuberization are discussed.     

Studies on the carbon dioxide promotion and ethylene inhibition of tuberization in potato explants cultured in vitro

Ethylene inhibited the tuberization of etiolated potato (Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro. Carbon dioxide did not overcome the C₂H₄ inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C₂H₄ prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin-increased tuberization of sprout sections.     

High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.     

Changes in the microtubular cytoskeleton precede in vitro tuber formation in potato

Summary An in vitro system for tuber formation was used to study early morphological and cytological changes occurring during tuber formation in potatoes, with special emphasis on the orientation of the microtubular cytoskeleton, visualized immunocytochemically. Axillary buds from potato plants were cultured in the presence or absence of gibberellin (GA), resulting in either tuber formation (without GA) or shoot formation (GA added). Tuber formation in the absence of GA was highly synchronous in individual buds, enabling the dissection of various aspects of tuberization. Under both conditions, starch started to accumulate. In the absence of GA, starch levels rapidly increased, concomitantly with tuber formation, whereas it slightly decreased in the presence of GA. Up to 4 days, the cortical MTs in the cells were oriented perpendicular to the longitudinal axis of the developing buds. Under tuber-inducing conditions this orientation changed into a longitudinal one at day 5. This change preceded a change in the direction of cell expansion. In the presence of GA no such reorientation was observed, cells continued to grow longitudinally, and a stoloniferous shoot was formed. The cytoskeletal changes preceded the visible swelling of the buds, observed after day 5, demonstrating that the reorientation of the microtubular cytoskeleton is one of the earliest steps observed so far in tuber formation in potatoes.Abbreviations GA gibberellin - MTs microtubules - PBS phosphate buffered saline - SD short-day     

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.     

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

Summary Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.