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Classical methods for the determination of polyamines in Arabidopsis, based on high-performance liquid chromatography or thin-layer chromatography, do not distinguish between aliphatic tetraamines or do not allow accurate quantitation, respectively. Here we describe a gas chromatography-mass spectrometry method that allows separation and simple quantitation of spermine and thermospermine. The reliability of the protocol has been confirmed using extracts of Arabidopsis mutants impaired in tetraamine biosynthesis. The levels of thermospermine are around 6-fold lower than those of spermine in Arabidopsis seedlings. Interestingly, hormonal effects on polyamine levels are not always correlated with those of transcripts of their biosynthetic enzymes., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Plants have long been considered advantageous platforms for large-scale production of antibodies due to their low cost, scalability, and the low chances of pathogen contamination. Much effort has therefore been devoted to efficiently producing mAbs (from nanobodies to secretory antibodies) in plant cells. Several technical difficulties have been encountered and are being overcome. Improvements in production levels have been achieved by manipulation of gene expression and, more efficiently, of cell targeting and protein folding and assembly. Differences in mAb glycosylation patterns between animal and plant cells are being successfully addressed by the elimination and introduction of the appropriate enzyme activities in plant cells. Another relevant battlefield is the dichotomy between production capacity and speed. Classically, stably transformed plant lines have been proposed for large scale mAb production, whereas the use of transient expression systems has always provided production speed at the cost of scalability. However, recent advances in transient expression techniques have brought impressive yield improvements, turning speed and scalability into highly compatible assets. In the era of personalized medicines, the combination of yield and speed, and the advances in glyco-engineering have made the plant cell a serious contender in the field of recombinant antibody production.

Fruit development is usually triggered by ovule fertilization, and it requires coordination between seed development and the growth and differentiation of the ovary to host the seeds. Hormones are known to synchronize these two processes, but the role of each hormone, and the mechanism by which they interact, are still unknown. Here we show that auxin and gibberellins (GAs) act in a hierarchical scheme. The synthetic reporter construct DR5:GFP showed that fertilization triggered an increase in auxin response in the ovules, which could be mimicked by blocking polar auxin transport. As the application of GAs did not affect auxin response, the most likely sequence of events after fertilization involves auxin-mediated activation of GA synthesis. We have confirmed this, and have shown that GA biosynthesis upon fertilization is localized specifically in the fertilized ovules. Furthermore, auxin treatment caused changes in the expression of GA biosynthetic genes similar to those triggered by fertilization, and also restricted to the ovules. Finally, GA signaling was activated in ovules and valves, as shown by the rapid downregulation of the fusion protein RGA-GFP after pollination and auxin treatment. Taken together, this evidence suggests a model in which fertilization would trigger an auxin-mediated promotion of GA synthesis specifically in the ovule. The GAs synthesized in the ovules would be then transported to the valves to promote GA signaling and thus coordinate growth of the silique.

As sessile organisms, plants modulate their growth rate and development according to the continuous variation in the conditions of their surrounding environment, an ability referred to as plasticity. This ability relies on a web of interactions between signaling pathways triggered by endogenous and environmental cues. How changes in environmental factors are interpreted by the plant in terms of developmental or growth cues or, in other words, how they contribute to plant plasticity is a current, major question in plant biology. Light stands out among the environmental factors that shape plant development. Plants have evolved systems that allow them to monitor both quantitative and qualitative differences in the light that they perceive, that render important changes in their growth habit. In this review we focus on recent findings about how information from this environmental cue is integrated during de-etiolation and in the shade-avoidance syndrome, and modulated by several hormone pathways-the endogenous cues. In some cases the interaction between a hormone and the light signaling pathways is reciprocal, as is the case of the gibberellin pathway, whereas in other cases hormone pathways act downstream of the environmental cue to regulate growth. Moreover, the circadian clock adds an additional layer of regulation, which has been proposed to integrate the information provided by light with that provided by hormone pathways, to regulate daily growth.

Polyamine biosynthesis is an ancient metabolic pathway present in all organisms. Aminopropyltransferases are key enzymes that mediate the synthesis of spermidine, spermine, and thermospermine. The relatively high sequence similarity between aminopropyltransferases and their similarity with putrescine N-methyltransferases (PMT) raises the question of whether they share a common ancestor or have evolved by convergence. Here we show that aminopropyltransferases and PMT are phylogenetically interconnected, and the different activities have been generated by unusually frequent events of diversification of existing functions. Although all spermidine synthases (SPDSs) derive from a common ancestor preceding the separation between prokaryotes and eukaryotes, they have been the origin of a variety of new activities. Among those, spermine synthases (SPMSs) represent a novelty independently arisen at least 3 times, in animals, fungi, and plants. The most parsimonious mechanism would involve the duplication and change of function of preexisting SPDS genes in each phylum. Although spermine is not essential for life, the repeated invention of SPMS and its conservation strongly argues for an evolutionary advantage derived from its presence. Moreover, the appearance of thermospermine synthase (tSPMS) in several genera of Archaea and Bacteria was accompanied by a loss of SPDS, suggesting that the new activity originated as a change of function of this enzyme. Surprisingly, tSPMS was later acquired by plants at an early stage of evolution by horizontal gene transfer and has proven to be essential for vascular development in tracheophytes. Finally, the synthesis of nicotine and tropane alkaloids in Solanales was favored by the origination of a new activity, PMT, as a duplication and change of function from SPDS.

In many plants, photomorphogenesis is the default developmental program after seed germination, and provides the key features that allow adaptation to light. This program is actively repressed if germination occurs in the absence of light, through a mechanism dependent on the E3 ubiquitin ligase activity that is encoded in Arabidopsis by COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), which induces proteolytic degradation of transcription factors necessary for light-regulated development, such as HY5 (LONG HYPOCOTYL 5) and HYH (LONG HYPOCOTYL 5 HOMOLOG), and stabilization of transcription factors that promote skotomorphogenesis, such as PIF3 (PHYTOCHROME INTERACTING FACTOR 3). Seedlings deficient in gibberellin (GA) synthesis or signaling display a de-etiolated phenotype when grown in darkness, equivalent to the phenotype of cop1 mutants, which indicates that the switch between photo- and skotomorphogenesis is also under hormonal control. Here we provide evidence for the existence of crosstalk between GA and the COP1-mediated pathway, and identify HY5 and the PIF family as nodes of a regulatory network. This interaction occurs through distinct molecular mechanisms, based on the observation that GA signaling regulates protein stability of HY5, and the activity of PIF3.

The formation of flowers involves the activity of a genetic network that acts in meristems to specify floral identity. The main output of this network is the initiation of a developmental patterning program for the generation of floral organs. The first characteristic of meristem identity genes is their capacity to integrate the environmental and endogenous cues that regulate the onset of flowering. This mechanism synchronizes temporal and spatial information, ensuring that flowers arise in the correct location at the appropriate time. The second characteristic of this network is the mutual regulatory interactions established between meristem identity genes. These interactions provide flexibility and robustness against environmental noise and prevent reversion once the decision to flower has been made. Finally, the third feature is the overlap between the meristem identity and other developmental programs that operate simultaneously to regulate different aspects of the construction of flowers.