The Role of CLE Peptides in the Suppression of Mycorrhizal Colonization of Tomato.

Symbioses with beneficial microbes are widespread in plants, but these relationships must balance the energy invested by the plants with the nutrients acquired. Symbiosis with arbuscular mycorrhizal (AM) fungi occurs throughout land plants, but our understanding of the genes and signals that regulate colonization levels is limited, especially in non-legumes. Here, we demonstrate that in tomato, two CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides, SlCLE10 and SlCLE11, act to suppress AM colonization of roots. Mutant studies and overexpression via hairy transformation indicate that SlCLE11 acts locally in the root to limit AM colonization. Indeed, SlCLE11 expression is strongly induced in AM-colonized roots, but SlCLE11 is not required for phosphate suppression of AM colonization. SlCLE11 requires the FIN gene that encodes an enzyme required for CLE peptide arabinosylation to suppress mycorrhizal colonization. However, SlCLE11 suppression of AM does not require two CLE receptors with roles in regulating AM colonization, SlFAB (CLAVATA1 ortholog) or SlCLV2. Indeed, multiple parallel pathways appear to suppress mycorrhizal colonization in tomato, as double mutant studies indicate that SlCLV2 and FIN have an additive influence on mycorrhizal colonization. SlCLE10 appears to play a more minor or redundant role, as cle10 mutants did not influence intraradical AM colonization. However, the fact that cle10 mutants had an elevated number of hyphopodia and that ectopic overexpression of SlCLE10 did suppress mycorrhizal colonization suggests that SlCLE10 may also play a role in suppressing AM colonization. Our findings show that CLE peptides regulate AM colonization in tomato and at least SlCLE11 likely requires arabinosylation for activity.

The influence of ethylene, gibberellins and brassinosteroids on energy and nitrogen-fixation metabolites in nodule tissue.

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome.

Brassinosteroids play multiple roles in nodulation of pea via interactions with ethylene and auxin.

MAIN CONCLUSION: A comprehensive analysis of the role of brassinosteroids in nodulation, including their interactions with auxin and ethylene revealed that brassinosteroids inhibit infection, promote nodule initiation but do not influence nodule organogenesis or function. Nodulation, the symbiosis between legumes and rhizobial bacteria, is regulated by a suite of hormones including brassinosteroids. Previous studies have found that brassinosteroids promote nodule number by inhibiting ethylene biosynthesis. In this study, we examined the influence of brassinosteroids on the various stages of infection and nodule development. We utilise pea mutants, including brassinosteroid mutants lk, lka and lkb, the ethylene insensitive ein2 mutant and the lk ein2 double mutant, along with transgenic lines expressing the DR5::GUS auxin activity marker to investigate how brassinosteroids interact with ethylene and auxin during nodulation. We show that brassinosteroids inhibit the early stages of nodulation, including auxin accumulation, root hair deformation and infection thread formation, and demonstrate that infection thread formation is regulated by brassinosteroids in an ethylene independent manner. In contrast, brassinosteroids appear to act as promoters of nodule initiation through both an ethylene dependent and independent pathway. Although brassinosteroids positively influence the ultimate number of nodules formed, we found that brassinosteroid-deficiency did not influence nodule structure including the vascular pattern of auxin activity or nitrogen-fixation capacity. These findings suggest that brassinosteroids are negative regulators of infection but positive regulators of nodule initiation. Furthermore, brassinosteroids do not appear to be essential for nodule organogenesis or function. Given the influence of brassinosteroids on discreet stages of nodulation but not nodule function, manipulation of brassinosteroids may be an interesting avenue for future research on the optimisation of nodulation.

Rht18 Semidwarfism in Wheat Is Due to Increased GA 2-oxidaseA9 Expression and Reduced GA Content.

Semidwarfing genes have improved crop yield by reducing height, improving lodging resistance, and allowing plants to allocate more assimilates to grain growth. In wheat (Triticum aestivum), the Rht18 semidwarfing gene was identified and deployed in durum wheat before it was transferred into bread wheat, where it was shown to have agronomic potential. Rht18, a dominant and gibberellin (GA) responsive mutant, is genetically and functionally distinct from the widely used GA-insensitive semidwarfing genes Rht-B1b and Rht-D1b In this study, the Rht18 gene was identified by mutagenizing the semidwarf durum cultivar Icaro (Rht18) and generating mutants with a range of tall phenotypes. Isolating and sequencing chromosome 6A of these "overgrowth" mutants showed that they contained independent mutations in the coding region of GA2oxA9GA2oxA9 is predicted to encode a GA 2-oxidase that metabolizes GA biosynthetic intermediates into inactive products, effectively reducing the amount of bioactive GA (GA1). Functional analysis of the GA2oxA9 protein demonstrated that GA2oxA9 converts the intermediate GA12 to the inactive metabolite GA110 Furthermore, Rht18 showed higher expression of GA2oxA9 and lower GA content compared with its tall parent. These data indicate that the increased expression of GA2oxA9 in Rht18 results in a reduction of both bioactive GA content and plant height. This study describes a height-reducing mechanism that can generate new genetic diversity for semidwarfism in wheat by combining increased expression with mutations of specific amino acid residues in GA2oxA9.

Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea.

The regulation of arbuscular mycorrhizal development and nodulation involves complex interactions between the plant and its microbial symbionts. In this study, we use the recently identified ethylene-insensitive ein2 mutant in pea (Pisum sativum L.) to explore the role of ethylene in the development of these symbioses. We show that ethylene acts as a strong negative regulator of nodulation, confirming reports in other legumes. Minor changes in gibberellin1 and indole-3-acetic acid levels in ein2 roots appear insufficient to explain the differences in nodulation. Double mutants produced by crosses between ein2 and the severely gibberellin-deficient na and brassinosteroid-deficient lk mutants showed increased nodule numbers and reduced nodule spacing compared with the na and lk single mutants, but nodule numbers and spacing were typical of ein2 plants, suggesting that the reduced number of nodules innaandlkplants is largely due to the elevated ethylene levels previously reported in these mutants. We show that ethylene can also negatively regulate mycorrhizae development when ethylene levels are elevated above basal levels, consistent with a role for ethylene in reducing symbiotic development under stressful conditions. In contrast to the hormone interactions in nodulation, ein2 does not override the effect of lk or na on the development of arbuscular mycorrhizae, suggesting that brassinosteroids and gibberellins influence this process largely independently of ethylene.

The role of strigolactones and ethylene in disease caused by Pythium irregulare.

Plant hormones play key roles in defence against pathogen attack. Recent work has begun to extend this role to encompass not just the traditional disease/stress hormones, such as ethylene, but also growth-promoting hormones. Strigolactones (SLs) are the most recently defined group of plant hormones with important roles in plant-microbe interactions, as well as aspects of plant growth and development, although the knowledge of their role in plant-pathogen interactions is extremely limited. The oomycete Pythium irregulare is a poorly controlled pathogen of many crops. Previous work has indicated an important role for ethylene in defence against this oomycete. We examined the role of ethylene and SLs in response to this pathogen in pea (Pisum sativum L.) at the molecular and whole-plant levels using a set of well-characterized hormone mutants, including an ethylene-insensitive ein2 mutant and SL-deficient and insensitive mutants. We identified a key role for ethylene signalling in specific cell types that reduces pathogen invasion, extending the work carried out in other species. However, we found no evidence that SL biosynthesis or response influences the interaction of pea with P. irregulare or that synthetic SL influences the growth or hyphal branching of the oomycete in vitro. Future work should seek to extend our understanding of the role of SLs in other plant interactions, including with other fungal, bacterial and viral pathogens, nematodes and insect pests.

Ethylene Signaling Influences Light-Regulated Development in Pea.

Plant responses to light involve a complex network of interactions among multiple plant hormones. In a screen for mutants showing altered photomorphogenesis under red light, we identified a mutant with dramatically enhanced leaf expansion and delayed petal senescence. We show that this mutant exhibits reduced sensitivity to ethylene and carries a nonsense mutation in the single pea (Pisum sativum) ortholog of the ethylene signaling gene ETHYLENE INSENSITIVE2 (EIN2). Consistent with this observation, the ein2 mutation rescues the previously described effects of ethylene overproduction in mature phytochrome-deficient plants. In seedlings, ein2 confers a marked increase in leaf expansion under monochromatic red, far-red, or blue light, and interaction with phytochromeA, phytochromeB, and long1 mutants confirms that ein2 enhances both phytochrome- and cryptochrome-dependent responses in a LONG1-dependent manner. In contrast, minimal effects of ein2 on seedling development in darkness or high-irradiance white light show that ethylene is not limiting for development under these conditions. These results indicate that ethylene signaling constrains leaf expansion during deetiolation in pea and provide further evidence that down-regulation of ethylene production may be an important component mechanism in the broader control of photomorphogenic development by phytochrome and cryptochrome.

Relationship between gibberellin, ethylene and nodulation in Pisum sativum.

• Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature. • Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development. • na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure. • Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis.

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.

A role for ethylene in the phytochrome-mediated control of vegetative development.

Members of the phytochrome family of photoreceptors play key roles in vegetative plant development, including the regulation of stem elongation, leaf development and chlorophyll accumulation. Hormones have been implicated in the control of these processes in de-etiolating seedlings. However, the mechanisms by which the phytochromes regulate vegetative development in more mature plants are less well understood. Pea (Pisum sativum) mutant plants lacking phytochromes A and B, the two phytochromes present in this species, develop severe defects later in development, including short, thick, distorted internodes and reduced leaf expansion, chlorophyll content and CAB gene transcript level. Studies presented here indicate that many of these defects in phyA phyB mutant plants appear to be due to elevated ethylene production, and suggest that an important role of the phytochromes in pea is to restrict ethylene production to a level that does not inhibit vegetative growth. Mutant phyA phyB plants produce significantly more ethylene than WT plants, and application of an ethylene biosynthesis inhibitor rescued many aspects of the phyA phyB mutant phenotype. This deregulation of ethylene production in phy-deficient plants appears likely to be due, at least in part, to the elevated transcript levels of key ethylene-biosynthesis genes. The phytochrome A photoreceptor appears to play a prominent role in the regulation of ethylene production, as phyA, but not phyB, single-mutant plants also exhibit a phenotype consistent with elevated ethylene production. Potential interactions between ethylene and secondary plant hormones in the control of the phy-deficient mutant phenotype were explored, revealing that ethylene may inhibit stem elongation in part by reducing gibberellin levels.