Comparative reproductive biology reveals two distinct pollination strategies in Neotropical twig-epiphyte orchids

Members of Oncidiinae are widely known for their interactions with oil-collecting bees that explore lipophilic secretions on flowers. They may also be pollinated through food deception and the offering of nectar. Although data on breeding systems are available for many Oncidiinae orchids, little is known about the reproductive strategies in Rodriguezia, a neotropical genus of ca. 55 species. In this paper, we explore the reproductive biology of two species of Rodriguezia with distinctive morphologies: R. decora and R. lanceolata. Floral features, spectral reflectance, pollinators and pollination mechanisms, and breeding systems were studied. Both species are scentless and produce nectar as a reward. Floral nectar is secreted by a gland at the base of the labellum and stored into the sepaline spur. Rodriguezia decora reflects mainly in the blue and red regions of the light spectrum, while R. lanceolata reflects in the red region. Rodriguezia decora is exclusively visited and pollinated by butterflies, while Trochilidae hummingbirds are the pollinators of R. lanceolata. Pollinaria attach to the upper third of the proboscis of butterflies (R. decora), and to the bill of hummingbirds (R. lanceolata), during the collection of nectar from the spur. Both Rodriguezia species are self-sterile. Flower features and floral reflectance support the occurrence of psychophily in R. decora and ornithophily in R. lanceolata.     

Variable domain I of nematode CLEs directs post-translational targeting of CLE peptides to the extracellular space

Effector proteins expressed in the esophageal gland cells of cyst nematodes are delivered into plant cells through a hollow, protrusible stylet. Although evidence indicates that effector proteins function in the cytoplasm of the syncytium,^1–3 technical constraints have made it difficult to directly determine where nematode effector proteins are initially delivered: cytoplasm, extracellular space, or both. Recently, we demonstrated that soybean cyst nematode CLE (HgCLE) propeptides are delivered to the cytoplasm of syncytial cells. Genetic and biochemical analyses indicate that the variable domain (VD) sequence is then required for targeting cytoplasmically delivered nematode CLEs to the apoplast where they function as ligand mimics of endogenous plant CLE peptides.⁴ The fact that nematode CLEs are targeted through the gland cell secretory pathway and delivered as mature propeptides into plant cells makes it impossible for these proteins to be subsequently delivered to the extracellular space via co-translational translocation through the endoplasmic reticulum (ER) secretory pathway of the host cell. However, when expressed in transgenic plants, if the mature nematode CLE propeptide harbored a functional cryptic signal peptide, it could possibly traffic to the apoplast through the ER secretory pathway by co-translational translocation. Here, we present evidence that VDI, the N-terminal sequence of the VD of HgCLE2,⁴ is sufficient for trafficking CLE peptides to the apoplast and that trafficking is indeed through an alternative pathway other than co-translational translocation.Key words: cyst nematode, effector, CLE, variable domain, trafficking, endoplasmic reticulum, co-translational translocation, post-translational     

A taxonomic revision of the Antennaria rosea (Asteraceae: Inuleae: Gnaphaliinae) polyploid complex

TheAntennaria rosea species complex is circumscribed to contain four subspecific taxa. A complete synonymy and key to the subspecies ofA. rosea is presented. The following new combinations are proposed:Antennaria rosea subsp.arida (E. Nels.) R. Bayer,A. rosea subsp. confinis (Greene) R. Bayer, andA. rosea subsp. pulvinata (Greene) R. Bayer. The affinities ofA. rosea to other species ofAntennaria are discussed and a key to separateA. rosea from related polyploid complexes is provided.     

Rice GH3 gene family: Regulators of growth and development

Auxin is an indispensable hormone throughout the lifetime of nearly all plant species. Several aspects of plant growth and development are rigidly governed by auxin, from micro to macro hierarchies; auxin also has a close relationship with plant-pathogen interactions. Undoubtedly, precise auxin levels are vitally important to plants, which have many effective mechanisms to maintain auxin homeostasis. One mechanism is conjugating amino acid to excessive indole-3-acetic acid (IAA; main form of auxin) through some GH3 family proteins to inactivate it. Our previous study demonstrated that GH3-2 mediated broad-spectrum resistance in rice (Oryza sativa L.) by suppressing pathogen-induced IAA accumulation and downregulating auxin signaling. Here, we further investigated the expression pattern of GH3-2 and other GH3 family paralogs in the life cycle of rice and presented the possible function of GH3-2 on rice root development by histochemical analysis of GH3-2 promoter:GUS reporter transgenic plants.Key words: auxin, GH3 gene, indole-3-acetic acid, Oryza sativa, rootThe phytohormone auxin regulates tropism and organ development and influences phyllotaxis, vascular canalization and root patterning by exerting its effect on cell division, elongation and differentiation in plants.^1,2 Indole-3-acetic acid (IAA) is the most widespread form of auxin in most plants. Supraoptimal or insufficient concentration of auxin will cause plants to exhibit abnormal phenotypes. 3-9 Auxin homeostasis is partly sustained by the GH3 gene family, a supervisor of the fluctuation of auxin. Most GH3 genes contain auxin-responsive cis-acting elements (AuxRE) in their promoter regions and react rapidly and transiently to auxin signaling.¹ Nineteen GH3 paralogs have been discovered in Arabidopsis.¹⁰ According to the phylogenetic relationship and acyl acid substrate preference, these genes are classified into three groups (I, II and III), which catalyze the formation of jasmonates, salicylic acid, 4-substituted benzoates or IAA acyl acid amido conjugates.^11,12 The rice GH3 gene family includes 13 paralogs, 4 belonging to group I (GH3-3, -5, -6 and -12) and 9 to group II (GH3-1, -2, -4, -7, -8, -9, -10, -11 and -13); group III GH3 is absent in rice.¹⁰ Rice GH3-1, -2, -8 and -13 paralogs have been biochemically confirmed to have IAA-amido synthetase activity by in vivo or in vitro assays.^6–9 It is believed that other GH3 group II paralogs in rice may also possess this enzymatic activity. But why does rice have such a functionally redundant group of GH3 proteins, which disobeys the economic principle? The explanation could be based on the different temporal and spatial expression of the genes encoding these proteins.     

The R1162 Mob Proteins Can Promote Conjugative Transfer from Cryptic Origins in the Bacterial Chromosome

The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in Pectobacterium atrosepticum, is shown here. A second active site was identified in Escherichia coli, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.The discovery over 50 years ago of bacterial mating by Lederberg and Cavalli-Sforza (summarized by Cavalli-Sforza [10]) was a major step in the development of modern microbial genetics. It was later realized that chromosomal DNA transfer was due to the integration of a plasmid, the F factor. This plasmid is able to effect its own transfer and, in the integrated state, chromosomal DNA as well. A unique site on the plasmid, the origin of transfer (oriT), is essential for this process. A complex of plasmid- and host-encoded proteins assemble at oriT (15); the F-encoded relaxase then cleaves one of the DNA strands, forming a covalent, protein-DNA intermediate (27) that is delivered to the type IV secretion apparatus, also encoded by F (24). The possibility of low-level transfer initiating from chromosomal sites was raised early in the history of conjugation (12). However, it is now clear that the exacting architecture of the oriT DNA-protein complex, both for the F factor and related oriTs, results in a very high degree of sequence and structural specificity, and no secondary origins in the chromosome have been identified.The broad-host-range plasmid R1162 (RSF1010), like the F factor, is also transferred from cell to cell. Although the mechanism of transfer is similar for the two plasmids, the oriTs are structurally very different. In prior studies we have determined that the R1162 oriT is small, structurally simple, and able to accommodate base changes at different positions without a complete loss of function (5, 21). As a result of this relaxed specificity, the R1162 mobilization (Mob) proteins can activate the related but different oriT of another plasmid, pSC101 (28).The R1162 oriT is shown in Fig. ​Fig.1.1. The R1162 relaxase MobA interacts with the core region, highly conserved in the R1162 Mob family (5), and the adjacent, inner arm of the inverted repeat (23). The protein forms at nic a tyrosyl phosphodiester linkage with the 5′ end of the DNA strand (30), which is then unwound from its complement as the protein-DNA complex is passed into the recipient cell. Circular plasmid DNA is reformed by a reverse of the initial protein-DNA transesterification, with the relaxase now binding to the 3′ end of the transferred DNA. This binding requires the complete inverted repeat, which probably forms a hairpin loop to recreate the double-stranded character of the relaxase binding site on duplex DNA.Open in a separate windowFIG. 1.Base sequence of R1162 oriT on the nicked strand. The relaxase cleaves at nic; subsequent transfer is 5′ to 3′ so that most of oriT is transferred last. Below is the consensus sequence of DNA active for initiation of transfer.An important feature of the steps in DNA processing at the R1162 oriT is that an inverted repeat is not required for the initiation of transfer or passage of DNA into a new cell. As a result of this and the permissiveness of the relaxase to base changes within oriT, different sites in the chromosome that are not part of a plasmid oriT might nevertheless be capable of initiating transfer when cloned into a plasmid. We previously identified one such site in the chromosome of Erwinia carotovora subsp. atroseptica (now Pectobacterium atrosepticum) (21). In addition, by testing libraries of oriTs with one or more mutations for relaxase-induced nicking, we found that a large population of DNAs with different sequences was potentially active (21). The consensus sequence for activity, derived from these studies, is shown in Fig. ​Fig.11.I show here that ectopic sites on the bacterial chromosome, active for the initiation of transfer of plasmid DNA, can also serve for the transfer of chromosomal DNA when the Mob proteins are provided in trans. Thus, the presence of R1162 in the cell can result in a cryptic sexuality in bacteria, resulting in the transfer of chromosomal genes. Although such events are likely to be infrequent, the broad-host-range of R1162 and its close relatives, with their ability to reside stably in the cytoplasms of many different bacteria, could be another source of gene exchange for a variety of different species.     

International plant molecular biology: a bright future for green science

A new study in this issue of Genome Biology sheds light on why some pseudogenes persist in rodent, and other mammalian, genomes. Please see related Research article by Marques et al http://genomebiology.com/2012/13/11/R102     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Walnut husk fly, <Emphasis Type="Italic">Rhagoletis completa</Emphasis> (Diptera: Tephritidae), invades Europe: invasion potential and control strategies

Rhagoletis completa Cresson (Diptera: Tephritidae) is native to North America and invaded Western Europe in the late 1980s, causing important damage to its principal host, walnut (Juglans spp.). In this review, we summarize the important elements of R. completa’s biology, phytosanitary status and methods used in Europe for its control, and then present the main conclusions associated with a completed risk analysis performed in 2014 to evaluate the dispersion and establishment potential of R. completa in Europe. The walnut husk fly was initially identified in Switzerland (1988) and Italy (1991), from where it spread to at least seven additional countries: France, Spain, Germany, Austria, Croatia, Slovenia and Hungary. R. completa has not reached the limits of its potential distribution. The main dissemination pathways within Europe include: (1) natural adult dissemination; (2) adult hitchhiker behaviour; and, to a lesser extent, (3) transportation of larvae in fresh fruits. R. completa host plants are widely distributed in Europe, either as isolated wild trees or in orchards, favouring the probability of fly establishment in currently fly-free areas. In addition, the European territories where Juglans species are present share biogeographic similarities. In orchards where R. completa is present and uncontrolled, 100% of walnut trees can be infested, causing losses in walnut yields of up to 80%. The negative effect is low (<10% yield loss) under phytosanitary control, although additional costs must also be considered to support specific monitoring for R. completa. The information presented here underlines a strong need for better walnut husk fly monitoring across European countries, as well as for increasing efforts to develop biological methods to control this emerging pest.     

The involvement of the Arabidopsis CRT1 ATPase family in disease resistance protein-mediated signaling

Resistance (R) gene-mediated immunity provides plants with rapid and strain-specific protection against pathogen infection. Our recent study using the genetically tractable Arabidopsis and turnip crinkle virus (TCV) pathosystem revealed a novel component, named CRT1 (compromised for recognition of the TCV CP), that is involved in general R gene-mediated signaling, including that mediated by HRT, an R gene against TCV. The Arabidopsis CRT1 gene family contains six additional members, of which two share high homology to CRT1 (75 and 81% a.a. identity); either CRT1 or its closest homolog restore the cell death phenotype suppressed by crt1. Analysis of single knock-out mutants for CRT1 and its closest homologs suggest that each may have unique and redundant functions. Here, we provide insight into the screening conditions that enabled identification of a mutant gene despite the presence of functionally redundant family members. We also discuss a potential mechanism that may regulate the interaction between CRT1 and R proteins.Key words: resistance gene, ATPase, suppressor screening, Arabidopsis, turnip crinkle virusPlant resistance (R) proteins activate defense signaling pathways following detection of a specific pathogen-encoded effector, or perception that a host factor has been altered by a pathogen effector. The vast majority of R proteins contain nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains. These R proteins can be further divided into two subgroups, TIR-NBS-LRR and CC-NBS-LRR, depending on whether the N terminus consists of a Toll-interleukin 1 receptor (TIR) or a coiled-coiled (CC) domain, respectively.¹ Subsequent to pathogen perception, the signal(s) generated by various R proteins likely converge into a limited set of pathways, with CC-NBS-LRR proteins usually utilizing NDR1 and TIR-NBS-LRR proteins generally requiring EDS1.² However, the molecular mechanism(s) through which R proteins recognize a pathogen(s) and initiate a defense signal(s) remains unclear.To gain insights into this elusive signaling process, several groups have performed genetic screens to isolate mutants whose R gene-mediated resistance responses are suppressed following either pathogen infection or expression of a transgene-encoded bacterial effector protein. Several proteins, including HSP90, SGT1 and RAR1, were shown to be required for resistance triggered by a variety of R proteins, suggesting their universal function in R protein-mediated resistance.^3–7 However, while some R protein-mediated signaling pathways required both RAR1 and SGT1, others needed only one or neither protein. Thus, the requirement for RAR1 and SGT1 appears to be specific to each pathway.⁸ Further studies revealed that SGT1, RAR1 and HSP90 regulate the stability/accumulation of various R proteins,^8–11 raising the possibility that they serve as (co)chaperones for assembling an active R protein complex.The Arabidopsis R protein HRT was previously shown to recognize the coat protein (CP) of turnip crinkle virus (TCV) and trigger necrotic lesion formation in the inoculated leaf, as well as local and systemic defense responses.¹² To identify components of the HRT-mediated signaling pathway, a line containing HRT and an inducible CP transgene was constructed and screened for suppressors of CP-induced cell death.¹³ One mutant, named crt1 (compromised for recognition of the TCV CP), was identified; it contains a mutation in a GHKL (Gyrase, Hsp90, histidine kinase, MutL) ATPase.¹³ Interestingly, HSP90 also belongs to this recently recognized ATPase superfamily, although sequence homology between HSP90 and CRT1 is limited to the ATPase domain.¹⁴ Either wt CRT1 or its closest homolog, CRT1-h1 (81% a.a. identity to CRT1; 13 suggesting that CRT1 and CRT1-h1 are functionally redundant.

Table 1

Amino-acid sequence identity between CRTI family members in Arabidopsis

Location Is Everything: An Educational Primer for Use with “Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae”

The article by Merwin et al. in the November 2014 issue of GENETICS provides insight into ribosome biogenesis, an essential multistep process that involves myriad factors and three cellular compartments. The specific protein of interest in this study is low-temperature viability protein (Ltv1), which functions as a small ribosomal subunit maturation factor. The authors investigated its possible additional function in small-subunit nuclear export. This Primer provides information for students to help them analyze the paper by Merwin et al. (2014), including an overview of the authors’ research question and methods.Related article in GENETICS: Merwin, J. R., L. B. Bogar, S. B. Poggi, R. M. Fitch, A. W. Johnson, and D. E. Lycan, 2014 Genetic analysis of the ribosome biogenesis factor Ltv1 of Saccharomyces cerevisiae. Genetics 198: 1071–1085     

Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile

Background

Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of ‘hypervirulent’ strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, two further CTns were described.

Results

CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.

Conclusions

The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.     

Leaf carbohydrate metabolism during defense: Intracellular sucrose-cleaving enzymes do not compensate repression of cell wall invertase

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.^1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. ³ and ⁴). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.^1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (K_m for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (K_m 7–15 mM).⁵Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. ^{3, 6–8}). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.^6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).¹¹ Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).^1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).¹Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.¹vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. ³ and ¹²). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,⁴ the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).¹ After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.¹ There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.^13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. ^{4, 14} and ¹⁵). Among other things they seem to be involved in osmotic or low-temperature stress response.^14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.^12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels¹⁷ but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.^1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.^1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.¹ After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.¹ It is known that expression of SUSY isoforms is differentially controlled by sugars,¹² and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.¹ In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.¹² The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation¹² and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell.