Publications - Cell and Metabolic Biology

Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant plants homozygous for T-DNA–disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair–specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.

The allene oxide cyclase (AOC) catalyzes the formation of cis-(+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the β-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.

Plants respond to mechanical wounding or herbivore attack with a complex scenario of sequential, antagonistic or synergistic action of different signals leading to defense gene expression. Tomato plants were used as a model system since the peptide systemin and the lipid-derived jasmonic acid (JA) were recognized as essential signals in wound-induced gene expression. In this review recent data are discussed with emphasis on wound-signaling in tomato. The following aspects are covered: (i) systemin signaling, (ii) JA biosynthesis and action, (iii) orchestration of various signals such as JA, H2O2, NO, and salicylate, (iv) local and systemic response, and (v) amplification in wound signaling. The common occurrence of JA biosynthesis and systemin generation in the vascular bundles suggest JA as the systemic signal. Grafting experiments with JA-deficient, JA-insensitive and systemin-insensitive mutants strongly support this assumption.

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199–213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.Barley plants contain one allene oxide cyclase and three allene oxide synthases which are up-regulated during seedling development accompanied by elevated levels of jasmonate.

Mycorrhizas are by far the most frequent occurring beneficial symbiotic interactions between plants and fungi. Species in >80% of extant plant families are capable of establishing an arbuscular mycorrhiza (AM). In relation to the development of the symbiosis the first molecular modifications are those associated with plant defense responses, which seem to be locally suppressed to levels compatible with symbiotic interaction (Gianinazzi-Pearson, 1996). AM symbiosis can, however, reduce root disease caused by several soil-borne pathogens. The mechanisms underlying this protective effect are still not well understood. In plants, products of the enzyme lipoxygenase (LOX) and the corresponding downstream enzymes, collectively named LOX pathway (Fig. 1B), are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity (Feussner and Wasternack, 2002). The central reaction in this pathway is catalyzed by LOXs leading to formation of either 9- or 13-hydroperoxy octadeca(di/trien)oic acids (9/13-HPO(D/T); Brash, 1999). Thus LOXs may be divided into 9- and 13-LOXs (Fig. 1A). Seven different reaction branches within this pathway can use these hydroperoxy polyenoic fatty acids (PUFAs) leading to (i) keto PUFAs by a LOX; (ii) epoxy hydroxy-fatty acids by an epoxy alcohol synthase (EAS); (iii) octadecanoids and jasmonates via allene oxide synthase (AOS); (iv) leaf aldehydes and leaf alcohols via fatty acid hydroperoxide lyase (HPL); (v) hydroxy PUFAs (reductase); (vi) divinyl ether PUFAs via divinyl ether synthase (DES); and (vii) epoxy- or dihydrodiolPUFAs via peroxygenase (PDX; Feussner and Wasternack, 2002). AOS, HPL and DES belong to one subfamily of P450-containing enzymes, the CYP74 family (Feussner and Wasternack, 2002). Here, the involvement of this CYP74 enzyme family in mycorrhizal roots of M. truncatula during early stages of AM symbiosis formation was analyzed.

Jasmonic acid (JA) and its precursor 12-oxo phytodienoic acid (OPDA) are lipid-derived signals in plant stress responses and development (Wasternack and Hause, 2002). Within the wound-response pathway of tomato, a local response of expression of defense genes such as the proteinase inhibitor 2 gene (PIN2) is preceded by a rise in JA (Herde et al., 1996; Howe et al., 1996) and ethylene (O’Donnell et al., 1996). Mutants affected in JA biosynthesis such as defl (Howe et al., 1996) or spr-2 (Li et al., 2002) clearly indicated that JA biosynthesis is an ultimate part of wound signaling. It is less understood, however, how the rise in JA is regulated.

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis (+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato.