Inflorescence stem grafting made easy in Arabidopsis

UNLABELLED BACKGROUND Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction. RESULTS Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem. CONCLUSIONS We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues

Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3' half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.Funding for this research is provided by the United States Department of Agriculture

Plant viral intergenic DNA sequence repeats with transcription enhancing activity

BACKGROUND: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection. RESULTS: DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components. CONCLUSION: Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function

A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis

A Spontaneous Dominant-Negative Mutation within a 35S::AtMYB90 Transgene Inhibits Flower Pigment Production in Tobacco

In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation.A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production.Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors

Simulated herbivory : the key to disentangling plant defence responses

Plants are subjected to a multitude of stimuli during insect herbivory, resulting in a complex and cumulative defence response. Breaking down the components of herbivory into specific stimuli and identifying the mechanisms of defence associated with them has thus far been challenging. Advances in our understanding of responses to inconspicuous stimuli, such as those induced by microbial symbionts in herbivore secretions and mechanical stimulation caused by insects, have illuminated the intricacies of herbivory. Here, we provide a synthesis of the interacting impacts of herbivory on plants and the consequential complexities associated with uncoupling defence responses. We propose that simulated herbivory should be used to complement true herbivory to decipher the mechanisms of insect herbivore-induced plant defence responses

A foliar pigment-based bioassay for interrogating chloroplast signalling revealed that carotenoid isomerisation regulates chlorophyll abundance

Background: Some plastid-derived metabolites can control nuclear gene expression, chloroplast biogenesis, and chlorophyll biosynthesis. For example, norfurazon (NFZ) induced inhibition of carotenoid biosynthesis in leaves elicits a protoporphyrin IX (Mg-ProtoIX) retrograde signal that controls chlorophyll biosynthesis and chloroplast development. Carotenoid cleavage products, known as apocarotenoids, also regulate plastid development. The key steps in carotenoid biosynthesis or catabolism that can regulate chlorophyll biosynthesis in leaf tissues remain unclear. Here, we established a foliar pigment-based bioassay using Arabidopsis rosette leaves to investigate plastid signalling processes in young expanding leaves comprising rapidly dividing and expanding cells containing active chloroplast biogenesis. Results: We demonstrate that environmental treatments (extended darkness and cold exposure) as well as chemical (norfurazon; NFZ) inhibition of carotenoid biosynthesis, reduce chlorophyll levels in young, but not older leaves of Arabidopsis. Mutants with disrupted xanthophyll accumulation, apocarotenoid phytohormone biosynthesis (abscisic acid and strigolactone), or enzymatic carotenoid cleavage, did not alter chlorophyll levels in young or old leaves. However, perturbations in acyclic cis-carotene biosynthesis revealed that disruption of CAROTENOID ISOMERASE (CRTISO), but not ZETA-CAROTENE ISOMERASE (Z-ISO) activity, reduced chlorophyll levels in young leaves of Arabidopsis plants. NFZ-induced inhibition of PHYTOENE DESATURASE (PDS) activity caused higher phytoene accumulation in younger crtiso leaves compared to WT indicating a continued substrate supply from the methylerythritol 4-phosphate (MEP) pathway. Conclusion: The Arabidopsis foliar pigment-based bioassay can be used to diferentiate signalling events elicited by environmental change, chemical treatment, and/or genetic perturbation, and determine how they control chloroplast biogenesis and chlorophyll biosynthesis. Genetic perturbations that impaired xanthophyll biosynthesis and/or carotenoid catabolism did not affect chlorophyll biosynthesis. The lack of CAROTENOID ISOMERISATION reduced chlorophyll accumulation, but not phytoene biosynthesis in young leaves of Arabidopsis plants growing under a long photoperiod. Findings generated using the newly customised foliar pigment-based bioassay implicate that carotenoid isomerase activity and NFZ-induced inhibition of PDS activity elicit different signalling pathways to control chlorophyll homeostasis in young leaves of Arabidopsis

Precise phenotyping for improved crop quality and management in protected cropping : a review

Protected cropping produces more food per land area than field-grown crops. Protected cropping includes low-tech polytunnels utilizing protective coverings, medium-tech facilities with some environmental control, and high-tech facilities such as fully automated glasshouses and indoor vertical farms. High crop productivity and quality are maintained by using environmental control systems and advanced precision phenotyping sensor technologies that were first developed for broadacre agricultural and can now be utilized for protected-cropping applications. This paper reviews the state of the global protected-cropping industry and current precision phenotyping methodology and technology that is used or can be used to advance crop productivity and quality in a protected growth environment. This review assesses various sensor technologies that can monitor and maintain microclimate parameters, as well as be used to assess plant productivity and produce quality. The adoption of precision phenotyping technologies is required for sustaining future food security and enhancing nutritional quality

Prior exposure of Arabidopsis seedlings to mechanical stress heightens jasmonic acid-mediated defense against necrotrophic pathogens

Background: Prolonged mechanical stress (MS) causes thigmomorphogenesis, a stress acclimation response associated with increased disease resistance. What remains unclear is if; 1) plants pre-exposed to a short period of repetitive MS can prime defence responses upon subsequent challenge with necrotrophic pathogens, 2) MS mediates plant immunity via jasmonic acid (JA) signalling, and 3) a short period of repetitive MS can cause long-term changes in gene expression resembling a stress-induced memory. To address these points, 10-days old juvenile Arabidopsis seedlings were mechanically stressed for 7-days using a soft brush and subsequently challenged with the necrotrophic pathogens, Alternaria brassicicola, and Botrytis cinerea. Here we assessed how MS impacted structural cell wall appositions, disease symptoms and altered gene expression in response to infection. Results: The MS-treated plants exhibited enhanced cell wall appositions and jasmonic acid (JA) accumulation that correlated with a reduction in disease progression compared to unstressed plants. The expression of genes involved in JA signalling, callose deposition, peroxidase and phytoalexin biosynthesis and reactive oxygen species detoxification were hyper-induced 4-days post-infection in MS-treated plants. The loss-of-function in JA signalling mediated by the JA-insensitive coronatine-insensitive 1 (coi1) mutant impaired the hyper-induction of defense gene expression and promoted pathogen proliferation in MS-treated plants subject to infection. The basal expression level of PATHOGENESIS-RELATED GENE 1 and PLANT DEFENSIN 1.2 defense marker genes were constitutively upregulated in rosette leaves for 5-days post-MS, as well as in naïve cauline leaves that differentiated from the inflorescence meristem well after ceasing MS. Conclusion: This study reveals that exposure of juvenile Arabidopsis plants to a short repetitive period of MS can alter gene expression and prime plant resistance upon subsequent challenge with necrotrophic pathogens via the JA-mediated COI1 signalling pathway. MS may facilitate a stress-induced memory to modulate the plant’s response to future stress encounters. These data advance our understanding of how MS primes plant immunity against necrotrophic pathogens and how that could be utilised in sustainable agricultural practices

Current technologies and target crops : a review on Australian protected cropping

Protected cropping offers a way to bolster food production in the face of climate change and deliver healthy food sustainably with fewer resources. However, to make this way of farming economically viable, we need to consider the status of protected cropping in the context of available technologies and corresponding target horticultural crops. This review outlines existing opportunities and challenges that must be addressed by ongoing research and innovation in this exciting but complex field in Australia. Indoor farm facilities are broadly categorised into the following three levels of technological advancement: low-, medium- and high-tech with corresponding challenges that require innovative solutions. Furthermore, limitations on indoor plant growth and protected cropping systems (e.g., high energy costs) have restricted the use of indoor agriculture to relatively few, high value crops. Hence, we need to develop new crop cultivars suitable for indoor agriculture that may differ from those required for open field production. In addition, protected cropping requires high start-up costs, expensive skilled labour, high energy consumption, and significant pest and disease management and quality control. Overall, protected cropping offers promising solutions for food security, while reducing the carbon footprint of food production. However, for indoor cropping production to have a substantial positive impact on global food security and nutritional security, the economical production of diverse crops will be essential