Search

Despite the agronomic importance of sugar beet (Beta vulgaris L.), the early‐stage development of its taproot has only been poorly investigated. Thus, the mechanisms that determine growth and sugar accumulation in sugar beet are largely unknown. In the presented study, a physiological characterization of early‐stage sugar beet taproot development was conducted. Activities were analyzed for fourteen key enzymes of carbohydrate metabolism in developing taproots over the first 80 days after sowing. In addition, we performed in situ localizations of selected carbohydrate‐metabolic enzyme activities, anatomical investigations, and quantifications of soluble carbohydrates, hexose phosphates, and phytohormones. Based on the accumulation dynamics of biomass and sucrose, as well as on anatomical parameters, the early phase of taproot development could be subdivided into three stages—prestorage, transition, secondary growth and sucrose accumulation stage—each of which was characterized by distinct metabolic and phytohormonal signatures. The enzyme activity signatures corresponding to these stages were also shown to be robustly reproducible in experiments conducted in two additional locations. The results from this physiological phenotyping approach contribute to the identification of the key regulators of sugar beet taproot development and open up new perspectives for sugar beet crop improvement concerning both physiological marker‐based breeding and biotechnological approaches.

Gene amplification followed by functional diversification is a major force in evolution. A typical example of this is seen in the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, named after the Arabidopsis stem cell regulator WUSCHEL. Here we analyze functional divergence in the WOX gene family. Members of the WUS clade, except the cambium stem cell regulator WOX4, can substitute for WUS function in shoot and floral stem cell maintenance to different degrees. Stem cell function of WUS requires a canonical WUS-box, essential for interaction with TPL/TPR co-repressors, whereas the repressive EAR domain is dispensable and the acidic domain seems only to be required for female fertility. In contrast to the WUS clade, members of the ancient WOX13 and the WOX9 clades cannot support stem cell maintenance. Although the homeodomains are interchangeable between WUS and WOX9 clade members, a WUS-compatible homeodomain together with canonical WUS-box is not sufficient for stem cell maintenance. Our results suggest that WOX function in shoot and floral meristems of Arabidopsis is restricted to the modern WUS clade, suggesting that stem cell control is a derived function. Yet undiscovered functional domains in addition to the homeodomain and the WUS-box are necessary for this function.

Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata, but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula, but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern.

Highlight The homeostasis of biologically active cytokinins was the predominant leaf-inherent factor for winter oilseed rape cultivar-differences in nitrogen starvation-induced leaf senescence and thus nitrogen efficiency., Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.

Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohormones have been demonstrated to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco, antagonistic interaction of these phytohormones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based on these results, we conclude that reduction of abscisic acid levels by enhanced abscisic acid catabolism strongly contributes to cytokinin-mediated resistance effects. Thus, the identified cytokinin–abscisic acid antagonism is a novel regulatory mechanism in plant immunity.

© The Author 2014. Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.

Phytoalexins are pathogen induced low molecular weight compounds with antimicrobial activities derived from secondary metabolism. Following their identification, phytoalexins were directly incorporated into the network of plant defense responses. Due to their heterogeneity, the metabolic pathways involved in phytoalexin formation and in particular the regulatory mechanisms remained elusive. Consequently, research focus shifted to the characterization of other components of plant immunity such as defense signaling and resistance mechanisms, including components of systemic acquired and induced systemic resistance, effector and pathogen-associated molecular pattern triggered immunity as well as R-gene resistance. Despite the obtained knowledge on these immunity mechanisms, genetic engineering employing these mechanisms and classical breeding reached too low improvements in crop protection, probably because classical breeding focused on yield performance and taste, rather than pathogen resistance. The increasing demand for disease resistant crop species and the aim to reduce pesticide application therefore requires alternative approaches. Recent advances in the understanding of phytoalexin function, biosynthesis and regulation, in combination with novel methods of molecular engineering and advances in instrumental analysis, returned attention to phytoalexins as a potent target for improving crop protection. Based on this, the advantages as well as potential bottlenecks for molecular approaches of modulating inducible phytoalexins to improve crop protection are discussed.

Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed.

The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance, crop yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric kinetic assays. The comparison of extraction buffers and requirement for dialysis of crude protein extracts resulted in a universal protein extraction protocol, suitable for the preparation of protein extracts from different organs of various species. Individual published kinetic activity assays were optimized and adapted for a semi-high-throughput 96-well assay format. These assays proved to be robust and are thus suitable for physiological phenotyping, enabling the characterization and diagnosis of the physiological state. The potential of the determination of distinct enzyme activity signatures as part of a physiological fingerprint was shown for various organs and tissues from three monocot and five dicot model and crop species, including two case studies with external stimuli. Differential and specific enzyme activity signatures are apparent during inflorescence development and upon in vitro cold treatment of young inflorescences in the monocot ryegrass, related to conditions for doubled haploid formation. Likewise, treatment of dicot spring oilseed rape with elevated CO2 concentration resulted in distinct patterns of enzyme activity responses in leaves.

A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network.

Gibberellic acid (GA) promotes germination, stem/hypocotyl elongation, and leaf expansion during seedling development. Using activation-tagging mutagenesis, we identified a mutation, sob2-D (for suppressor of phytochromeB-4 [phyB-4]#2 dominant), which suppresses the long-hypocotyl phenotype of a phyB missense allele, phyB-4. This mutant phenotype is caused by the overexpression of an APETALA2 transcription factor, SOB2, also called DRN-like. SOB2/DRN-like transcript is not detectable in wild-type seedling or adult tissues via RT-PCR analysis, suggesting that SOB2/DRN-like may not be involved in seedling development under normal conditions. Adult sob2-D phyB-4 plants have curled leaves and club-like siliques, resembling plants that overexpress a closely related gene, LEAFY PETIOLE (LEP). Hypocotyls of a LEP-null allele, lep-1, are shorter in the light and dark, suggesting LEP involvement in seedling development. This aberrant hypocotyl phenotype is due at least in part to a delay in germination. In addition, lep-1 is less responsive to GA and more sensitive to the GA biosynthesis inhibitor paclobutrazol, indicating that LEP is a positive regulator of GA-induced germination. RT-PCR shows that LEP transcript accumulates in wild-type seeds during imbibition and germination, and the transcript levels of REPRESSOR OF ga1-3-LIKE2 (RGL2), a negative regulator of GA signaling during germination, is unaffected in lep-1. These results suggest LEP is a positive regulator of GA-induced germination acting independently of RGL2. An alternative model places LEP downstream of RGL2 in the GA-signaling cascade.

Transgenic Arabidopsis thaliana plants harboring the Agrobacterium tumefaciens phytohormone-biosynthetic genes iaaM and iaaH display an altered development indicative of elevated auxin levels. These plants exhibit an increased apical dominance, increased hypocotyl and petiole length and epinastic leaves and cotyledons. In addition, the transgenic plants display the formation of necrotic spots on leaves and bracts in soil and an altered behavior of axillary buds in tissue culture. Despite transcriptional activity of the iaaM and iaaH promoters in the vasculature of root tissue, root development of young iaaM/iaaH transgenic plants is identical to wild type. However, transgenic iaaM/iaaH plants grown under tissue culture conditions for a prolonged period display both vigorous and ectopic root growth. The level of free IAA in the rosette of the iaaM/iaaH transgenic plants is increased approximately 2-fold compared to wild type. The number of cells comprising the vascular tissue in hypocotyls and inflorescence stems of the transgenic plants is reduced and these plants exhibit a reduced basipetal polar auxin transport in the inflorescence stems. This reduced polar auxin transport probably accounts for the rapid auxin effect on the aerial part of the transgenic plants and the late effect on root development.

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.

The complex dominant Arabidopsis thaliana mutant lettuce (let) shows the conversion of the leaf petiole into a leaf blade caused by an ectopic leaf blade formation. This is the result of the activation tagging of the LEAFY PETIOLE (LEP) gene encoding an AP2/EREBP-like transcription factor. Here, we report that in addition to this leafy petiole phenotype, the size of the vascular bundles is increased in all aerial organs in let as a result of an increase in the number of xylem, phloem (pro)cambial and pericycle cells. This vascular phenotype is caused by activation tagging of the two genes VASCULAR TISSUE SIZE (VAS) and LEP. These genes are closely linked and arranged in tandem. Activation tagging of LEP only caused a specific increase in the number of xylem cells. This increased xylem cell number, together with the ectopic leaf blade formation, indicates that LEP functions as a cell division-promoting factor. The activation tagging of VAS only resulted in a specific increase in phloem (pro)cambial and pericycle cells. We conclude that activation tagging of LEP and VAS results in additive phenotypes. Insertional mutants for LEP and VAS display wild-type vascular development, indicating the relevance of activation tagging for functional analysis of novel genes involved in plant development.

The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)-specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6-phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6-bisphosphatase. Despite its regulatory role in the feed-forward control of sucrose biosynthesis, variations in the fructose 2,6-bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt-1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1-1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt-1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady-state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.

The Arabidopsis thaliana (L.) Heynh. mutant delayed-dehiscence2-2 (dde2-2) was identified in an En1/Spm1 transposon-induced mutant population screened for plants showing defects in fertility. The dde2-2 mutant allele is defective in the anther dehiscence process and filament elongation and thus exhibits a male-sterile phenotype. The dde2-2 phenotype can be rescued by application of methyl jasmonate, indicating that the mutant is affected in jasmonic acid biosynthesis. The combination of genetic mapping and a candidate-gene approach identified a frameshift mutation in the ALLENE OXIDE SYNTHASE (AOS) gene, encoding one of the key enzymes of jasmonic acid biosynthesis. Expression analysis and genetic complementation of the dde2-2 phenotype by overexpression of the AOS coding sequence confirmed that the male-sterile phenotype is indeed caused by the mutation in the AOS gene.

Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty. Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

From a pool of transgenic Arabidopsis (Arabidopsis thaliana) plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D) on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD). The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD.

In response to stress, plants accumulate Pro, requiring degradation after release from adverse conditions. Δ1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death.

Phytohormones are essential regulators of various processes in plant growth and development. Several phytohormones are also known to regulate plant responses to environmental stress and pathogens. Only recently, cytokinins have been demonstrated to play an important role in plant immunity. Increased levels of cytokinins such as trans-zeatin, which are considered highly active, induced resistance against mainly (hemi)biotrophic pathogens in different plant species. In contrast, cis-zeatin is commonly regarded as a cytokinin exhibiting low or no activity. Here we comparatively study the impact of both zeatin isomers on the infection of Nicotiana tabacum by the (hemi)biotrophic microbial pathogen Pseudomonas syringae. We demonstrate a biological effect of cis-zeatin and a differential effect of the two zeatin isomers on symptom development, defense responses and bacterial multiplication.