Cell and Metabolic Biology

Calcium has been largely hypothesized to be involved in the arbuscular mycorrhizal (AM) signal transduction. A transient elevation in cytosolic Ca 2+ concentration is considered one of the earliest responses detected in plant cells challenged with different stimuli. In this paper the detection of Ca 2+ transients was selected as a tool to analyse whether plant cells perceive diffusible molecules released by the AM fungus Glomus intraradices. An aequorin-transformed line of soybean cells was used as experimental system. When cells were treated with the medium in which Gl. intraradices spores germinated, a rapid and significant increase in cytosolic Ca 2+ level was induced and dissipated within 30 min. The medium did not induce either intracellular accumulation of reactive oxygen species or cell death. A detectable Ca 2+ elevation was triggered also by the medium of fungal extraradical mycelium, associated to mycorrhizal hairy roots of carrot. These results indicate that Gl. intraradices releases diffusible molecules that are sensed by host plant cells through a Ca 2+ -mediated signalling during different steps of its life cycle.

We have searched for induced transcripts in a cDNA library derived from bean cell suspension cultures treated with an elicitor from Colletotrichum lindemuthianum. Six independently isolated cDNAs corresponding to rapidly induced small mRNAs have been classified by their DNA sequence and slightly different induction behaviour into two groups. 5'-and 3'-untranslated regions exhibit little similarity, but the deduced small acidic proteins designated PvPRI and PvPR2 are 89% identical. No relationship was found with the wellcharacterized PR1 proteins from tobacco. However, the PvPR proteins are closely related to pi49 in pea (64% identity), pSTH2 in potato (41% identity) and PcPRI-1 in parsley (39% identity), which are also induced in response to elicitor or microbial attack. Moreover, a major pollen allergen in birch (Betvl) has a 44% identity with PvPR1 proteins. These similarities establish a ubiquitous class of conserved defense-related proteins and suggest a common yet still unknown function. Southern blot analysis indicates that PvPR protein gene organization is highly complex with an estimated copy number of more than 12 genes.

This review focuses on plant carotenoids, but it also includes progress made on microbial and animal carotenoid metabolism to better understand the functions and the evolution of these structurally diverse compounds with a common backbone. Plants have evolved isogenes for specific key steps of carotenoid biosynthesis with differential expression profiles, whose characteristic features will be compared. Perhaps the most exciting progress has been made in studies of carotenoid cleavage products (apocarotenoids) with an ever-expanding variety of novel functions being discovered. This review therefore covers structural, molecular genetic and functional aspects of carotenoids and apocarotenoids alike. Apocarotenoids are specifically tailored from carotenoids by a family of oxidative cleavage enzymes, but whether there are contributions to their generation from chemical oxidation, photooxidation or other mechanisms is largely unknown. Control of carotenoid homeostasis is discussed in the context of biosynthetic and degradative reactions but also in the context of subcellular environments for deposition and sequestration within and outside of plastids. Other aspects of carotenoid research, including metabolic engineering and synthetic biology approaches, will only be covered briefly.

Cell suspension cultures of parsley (Petroselinum crispum) exhibited an altered pattern of protein synthesis after transfer from complete growth medium to water or medium containing no macronutrients. Similar changes occurred when cultures were grown in the original medium until the nutrients were depleted. The effect was reversible upon transfer to fresh medium and was not observed during regular subculturing of the cells. While total protein synthesis decreased sharply after nutrient depletion, the synthesis of a few characteristic proteins (starvation-related proteins, STPs) increased strongly. The protein labeled at highest rates with [3SS]methionine in vivo (STP 62) had an apparent molecular weight of about 62000 and a pI of about 6.3. Although its increased rate of synthesis was therefore easily detected by labeling in vivo, translation of mRNA in vitro did not give comparable results. Thus, regulatory control may be exerted mainly at the level of translation. Synthesis of STP ceased rapidly when heat shock (37 ~ C) was applied under conditions of nutrient depletion, whereas heat-shock proteins were strongly induced.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a Agtll library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence ofa second, divergent CAD gene involved in lignification during xylogenesis are discussed.

The intracellular pathogenesis-related (PR) proteins of common bean (Phnseolus vulgaris L.) are encoded by a highly polymorphic family of at least 20 genes. One member, the YprlO*c gene, has been isolated and characterised. The deduced amino acid sequence of the encoded protein, PR-10, exhibits similarities to tree-pollen allergens, to food allergens from celery and apple and to ginseng ribonuclease peptide sequences. We show by RNA blot analysis that the YprlO gene family, including YprlO*c, is strongly expressed in bean roots. In leaves YprlO transcript levels are low in young and mature stages but are elevated during senescence and in diseased states. Dark treatment of leaves causes strong induction of YprlO transcripts, which is reversible by light, and diurnal rhythms of transcript accumulation during the night are observed. YprlO genes are responsive to external stimuli related to pathogen-defence such as glutathione or salicylic acid. Transcriptional activity of a Ypr1O"c promoter-/j-glucuronidase fusion gene in transgenic tobacco was observed in roots, in developing xylem and phloem of stems, and in the blade of senescent leaves, with highest levels at the onset of senescence. The most striking characteristic of developmental expression was the specific localisation of /~-glucuronidase activity in the transmitting tract of styles in flowers at anthesis. Feeding of various pathogen-related and stress-related stimuli to young tobacco leaves led to accumulation of GUS activity in leaf blades. We identify considerable spatiotemporal similarities between reported expression patterns of YprlO genes and ribonuclease genes, which, together with the significant sequence similarity to the ginseng ribonuclease, support the hypothesis of a ribonuclease function for PR-10 proteins and allow the prediction of possible biological roles.

Plants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with speci®c and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C 13 cyclohexenone derivatives and mycorradicin (C 14 ) conjugates, the latter being a major component of the long-knowǹ yellow pigment'. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identi®ed from mycorrhizal wheat roots after alkaline treatment of an`apocarotenoid complex' of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the ®rst report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.

Oxidative tailoring of C(40) carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C(13) and C(14) apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C(40) substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C(27) apocarotenoids but not C(40) carotenoids suggesting C(27) as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C(40) to C(27) in the plastid followed by C(27) export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the...

The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase (DXS1 and DXS2). In Medicago truncatula, MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM-induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non-AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong re...