Cold hardening in conifers includes growth cessation and long-term changes in metabolism. In the conifers of the boreal forest (Fig. 1), this process is induced by short days and potentiated by low temperature. A problem encountered by overwintering evergreen conifers such as Pinus banksiana is that they retain a substantial amount of chlorophyll (Chl) throughout the winter and hence continue to absorb light, while at the same time there is a short photoperiod-induced, low temperature-induced, down-regulation of metabolism. Under such conditions, light capture and energy utilization must be regulated in a coordinated manner to prevent oxidative damage to the photosynthetic apparatus. Energy balance, defined as photostasis, is achieved by reorganization of the photosynthetic machinery, including changes in antenna size and organization, adjustments of protein and Chl concentrations, and a wide range of alternative energy dissipation pathways. The length of the growing season in the boreal forests is projected to increase by 20 to 30 d by 2080, thereby possibly improving the productivity of northern boreal forests. However, it has also been suggested that boreal conifers might be unable to fully exploit an extended growing season because prolonged warmer temperatures during autumn may interfere with cold hardening. The question of how global warming will affect photostasis in boreal pine forests is the topic of a contribution by Busch et al. (pp. 402–414). The authors show that a simulated increase in autumn air temperature inhibits CO₂ assimilation in P. banksiana and that this occurs concomitantly with an increase in nonphotochemical quenching of absorbed energy. Photoprotection under increased autumn air temperature conditions is consistent with zeaxanthin-independent antenna quenching through LHCII aggregation and a decreased efficiency in energy transfer from the antenna to the PSII core. Although it is difficult to extrapolate these findings with Pinus seedlings growing under stimulated conditions to mature forest stands, the results of these experiments demonstrate the significance of warm autumn temperatures on the cold-hardening process in conifers that warrant further investigation under more natural conditions.

In addition to their hallucinogenic properties, ergoline alkaloids are also the active ingredients in medications designed to treat migraines or Parkinson's disease. The occurrence of ergoline alkaloids in nature is oddly distributed: They occur in ascomycetes belonging to the genera Claviceps, Aspergillus, and Penicillium, in the dicotyledonous plant families Convolvulaceae and Polygalaceae, and have even been isolated from a tunicate animal. In the case of the Convolvulaceae, the treatment of Ipomoea asarifolia and Turbina corymbosa with fungicides leads to the removal of epibiotic fungi and a concomitant loss of ergoline alkaloids from the plants. The biosynthesis of ergoline alkaloids apparently depends on the presence of an intact host plant. An ergoline alkaloid-producing fungus is also present in I. asarifolia callus and cell suspension cultures that, however, contain no ergoline alkaloids. A plant regenerated from such a callus culture, however, does contain ergoline alkaloids and is colonized by the fungus. Markert et al. (pp. 296–305) present further evidence that fungi are responsible for ergoline alkaloid biosynthesis. The dmaW gene, which codes for a key enzyme in ergoline alkaloid biosynthesis, was found to be part of the epibiotic fungal genome. Neither the gene nor the biosynthetic capacity was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Ergoline alkaloids, however, were found to be stored almost exclusively in the plants and were not detectable in the associated epibiotic fungi. This indicates that a highly efficient transport system must be in place to translocate the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant is very likely a symbiosis in which ergoline alkaloids play an essential role.

About 40% of the proteins encoded in eukaryotic genomes are proteins of unknown function (PUFs). Two major operational definitions have been used to identify PUFs in model organisms. The similarity approach to protein function identification considers PUFs to be all proteins that show no sequence or structural similarities to functionally characterized proteins in reference databases. The more conservative empirical approach, on the other hand, defines PUFs as all proteins that lack direct experimental evidence as to their specific function. Important clues about the possible functions of PUFs can be garnered by studying the proteins of known function (PKFs) that are expressed in association with PUFs. In this issue, Horan et al. (pp. 41–57) report on their identification and genome-wide analysis of PUF encoding genes from Arabidopsis (Arabidopsis thaliana) using both empirical and similarity strategies. Large-scale analyses of publicly available gene expression array data allowed them to associate PUF with PKF genes based on similarities of their expression and treatment response profiles. Genome-wide clustering and gene function enrichment analysis of clusters allowed them to associate 1,541 PUF genes with tightly coexpressed PFK genes. More than 70% of PUFs could be assigned to more specific biological process annotations than the ones available in the current Gene Ontology release. The most highly overrepresented functional categories in the obtained clusters were ribosome assembly, photosynthesis, and cell wall pathways. Large-scale analyses of differentially expressed genes were also applied to identify a comprehensive set of abiotic stress response genes. This analysis resulted in the identification of 269 PKF and 104 PUF genes that responded to a wide variety of abiotic stresses, while 608 PKF and 206 PUF genes responded predominantly to specific stress treatments. Stemming from this project, the authors have also developed a public Plant Gene Expression Database to provide efficient access and mining tools for the vast gene expression data they have generated.

Crassulacean acid metabolism (CAM) is a mode of photosynthetic carbon fixation that has evolved by convergence in approximately 7% of vascular plant species. Efforts to elucidate the metabolic, regulatory, and signaling elements that define CAM have focused largely on the facultative CAM halophyte Mesembryanthemum crystallinum (common ice plant). When grown under well-watered, nonstressed conditions, M. crystallinum performs C₃ photosynthesis, but can switch to CAM if subjected to various treatments that reduce water availability, including high salinity. To date, genetic dissection of the regulatory and metabolic attributes of CAM in M. crystallinum has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Cushman et al. (pp. 228–238) report the establishment of large-scale M. crystallinum mutant collections and the development of a novel and simple screening strategy for the isolation of CAM-deficient mutants in M. crystallinum that employs a pH indicator to rapidly identify salt-stressed plants that fail to perform nocturnal acidification, a diagnostic characteristic of CAM. The isolated CAM-deficient mutants showed negligible net dark CO₂ uptake compared with wild-type plants following the imposition of salinity stress. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C₄ acid formation. The ability to accumulate and degrade sufficient starch reserves during the day to supply sufficient phosphoenolpyruvate for nocturnal C₄ carboxylation is a prerequisite of CAM in M. crystallinum. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Arabidopsis Suc transporter AtSUC1 is a proton-coupled Suc uptake transporter. AtSUC1 is expressed in pollen, trichomes, and roots. In pollen, AtSUC1 has been proposed to function in Suc uptake during germination. AtSUC1 mRNA accumulates during pollen maturation, but AtSUC1 protein is not detectable in pollen until germination. Sivitz et al. (pp. 92–100) tested the hypothesis that AtSUC1 functions in pollen germination using atsuc1 insertional mutants and show that atsuc1 mutant pollen is defective in germination both in vivo and in vitro. As expected, AtSUC1-GFP was localized to the plasma membrane in pollen tubes. AtSUC1 is also expressed in roots and the external application of Suc increased AtSUC1 expression in roots. The authors also report that AtSUC1 is important for Suc-dependent signaling leading to anthocyanin accumulation in cotyledons, especially around the edges, and at the tip of the hypocotyls. suc1 mutants accumulated less anthocyanins in response to exogenous Suc or maltose, and microarray analysis revealed the reduced expression of many genes important for anthocyanin biosynthesis. The authors hypothesize that Suc uptake is necessary for Suc-induced anthocyanin production and that other Suc transporters cannot compensate for the loss of AtSUC1 activity. These results indicate that AtSUC1 is important for sugar signaling in vegetative tissue and for normal male gametophyte function.

Transgenic plants can produce complex mammalian proteins requiring extensive posttranslational modifications. This ability makes plants an attractive alternative for the production of certain therapeutic proteins. Several studies have demonstrated that N-glycosylation is one of the most common and important posttranslational modifications of proteins, and an advantage of using plants as an expression system is their ability to perform N-glycosylation similar to mammalian cells. Previous studies, however, have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac). Castilho et al. (pp. 331–339) report the successful in planta expression of three enzymes required for the synthesis of CMP-Neu5Ac in mammals. The simultaneous expression of these genes in Arabidopsis resulted in the generation of high amounts of Neu5Ac and CMP-Neu5Ac from endogenous precursors. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.