Altered gene expression by sedaxane increases PSII efficiency, photosynthesis and growth and improves tolerance to drought in wheat seedlings

Succinate dehydrogenase inhibitor (SDHI) fungicides have been shown to increase PSII efficiency and photosynthesis under drought stress in the absence of disease to enhance the biomass and yield of winter wheat. However, the molecular mechanism of improved photosynthetic efficiency observed in SDHI-treated wheat has not been previously elucidated. Here we used a combination of chlorophyll fluorescence, gas exchange and gene expression analysis, to aid our understanding of the basis of the physiological responses of wheat seedlings under drought conditions to sedaxane, a novel SDHI seed treatment. We show that sedaxane increased the efficiency of PSII photochemistry, reduced non-photochemical quenching and improved the photosynthesis and biomass in wheat correlating with systemic changes in the expression of genes involved in defense, chlorophyll synthesis and cell wall modification. We applied a coexpression network-based approach using differentially expressed genes of leaves, roots and pregerminated seeds from our wheat array datasets to identify the most important hub genes, with top ranked correlation (higher gene association value and z-score) involved in cell wall expansion and strengthening, wax and pigment biosynthesis and defense. The results indicate that sedaxane confers tolerant responses of wheat plants grown under drought conditions by redirecting metabolites from defense/stress responses towards growth and adaptive development

Investigating the role of tetrapyrrole biosynthesis under drought stress in cereal transgenics

The tetrapyrrole biosynthesis pathway leads to chlorophyll and heme production and plays a key role in primary physiological processes such as photosynthesis and respiration. Recent studies have shed light on heme as a potential candidate molecule for triggering stress defence responses. However, detailed investigations are yet to be conducted to elucidate the potential role of heme in regulating responses to complex abiotic stress conditions such as drought. The terminal enzyme of heme biosynthesis is Ferrochelatase (FC), for which there are two isoforms encoded by separate genes (FC1 and FC2). Previous studies propose that the two FCs synthesize two physiologically distinct heme pools with different cellular functions. The overall scientific goal of this thesis was to investigate the roles of the two FCs in photosynthesis, drought and oxidative stress tolerance. In this study, barley (Hordeum vulgare) was used as both a major cereal crop and also as a model plant for other commercially relevant rain-fed cereal crops. Two FCs in barley (HvFC1 and HvFC2) were identified and their tissue-specific and stress-responsive expression patterns were investigated. These genes were cloned from the cultivar Golden Promise (GP) and transgenic lines ectopically overexpressing either HvFC1 or HvFC2 were generated. From 29 independent T₀ transgenic lines obtained for each FC construct, three single-copy transgenic lines ectopically overexpressing either HvFC1 or HvFC2 were evaluated for photosynthetic performance, oxidative and drought stress tolerance. The two HvFC isoforms share a common catalytic FC domain, while HvFC2 additionally contains C-terminal chlorophyll a/b binding (CAB) domain. The two genes are differentially expressed in photosynthetic and non-photosynthetic tissues and have distinct stress responsive expression profiles, implying that they may have distinct roles. Transgenic plants ectopically overexpressing either HvFC1 or HvFC2 exhibited significantly higher chlorophyll content, stomatal conductance (gs) [s subscript], carboxylation efficiency (CE) and photosynthetic rate relative to controls under both non-stressed and drought stress conditions. Furthermore, these transgenics, showed wilting avoidance and maintained higher leaf water content and water use efficiency relative to control plants when subjected to drought stress. Overexpression of HvFCs significantly up-regulated nuclear genes associated with ROS detoxification under drought stress. It also reduced photo-oxidative damage caused by perturbation of tetrapyrrole biosynthesis in tigrinaᵈ¹² mutants. Taken together, this study indicates that both HvFCs play roles in photosynthesis and improving oxidative and drought stress tolerance. The results reported in this thesis suggest that both HvFC derived heme pools are likely to be involved in chloroplast-to-nuclear retrograde signaling to trigger drought and oxidative stress tolerance. This study also highlights the tetrapyrrole pathway as an important target for engineering improved crop performance in both non-stressed and stressed environments.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food & Wine, 2015

Tetrapyrrole-based drought stress signalling

Tetrapyrroles such as chlorophyll and heme play a vital role in primary plant metabolic processes such as photosynthesis and respiration. Over the past decades, extensive genetic and molecular analyses have provided valuable insights into the complex regulatory network of the tetrapyrrole biosynthesis. However, tetrapyrroles are also implicated in abiotic stress tolerance, although the mechanisms are largely unknown. With recent reports demonstrating that modified tetrapyrrole biosynthesis in plants confers wilting avoidance, a component physiological trait to drought tolerance, it is now timely that this pathway be reviewed in the context of drought stress signalling. In this review, the significance of tetrapyrrole biosynthesis under drought stress is addressed, with particular emphasis on the inter-relationships with major stress signalling cascades driven by reactive oxygen species (ROS) and organellar retrograde signalling. We propose that unlike the chlorophyll branch, the heme branch of the pathway plays a key role in mediating intracellular drought stress signalling and stimulating ROS detoxification under drought stress. Determining how the tetrapyrrole biosynthetic pathway is involved in stress signalling provides an opportunity to identify gene targets for engineering drought-tolerant crops.Dilrukshi S.K. Nagahatenna, Peter Langridge and Ryan Whitfor

Altering tetrapyrrole biosynthesis by overexpressing ferrochelatases (Fc1 and Fc2) improves photosynthetic efficiency in transgenic barley

Ferrochelatase (FC) is the terminal enzyme of heme biosynthesis. In photosynthetic organisms studied so far, there is evidence for two FC isoforms, which are encoded by two genes (FC1 and FC2). Previous studies suggest that these two genes are required for the production of two physiologically distinct heme pools with only FC2-derived heme involved in photosynthesis. We characterised two FCs in barley (Hordeum vulgare L.). The two HvFC isoforms share a common catalytic domain, but HvFC2 additionally contains a C-terminal chlorophyll a/b binding (CAB) domain. Both HvFCs are highly expressed in photosynthetic tissues, with HvFC1 transcripts also being abundant in non-photosynthetic tissues. To determine whether these isoforms differentially affect photosynthesis, transgenic barley ectopically overexpressing HvFC1 and HvFC2 were generated and evaluated for photosynthetic performance. In each case, transgenics exhibited improved photosynthetic rate (Asat), stomatal conductance (gs) and carboxylation efficiency (CE), showing that both FC1 and FC2 play important roles in photosynthesis. Our finding that modified FC expression can improve photosynthesis up to ~13% under controlled growth conditions now requires further research to determine if this can be translated to improved yield performance under field conditions.Dilrukshi S. K. Nagahatenna, Jingwen Tiong, Everard J. Edwards, Peter Langridge and Ryan Whitfor

Barley Plants Overexpressing Ferrochelatases (HvFC1 and HvFC2) Show Improved Photosynthetic Rates and Have Reduced Photo-Oxidative Damage under Drought Stress than Non-Transgenic Controls

We investigated the roles of two Ferrochelatases (FCs), which encode the terminal enzyme for heme biosynthesis, in drought and oxidative stress tolerance in model cereal plant barley (Hordeum vulgare). Three independent transgenic lines ectopically overexpressing either barley FC1 or FC2 were selected and evaluated under well-watered, drought, and oxidative stress conditions. Both HvFC1 and HvFC2 overexpressing transgenics showed delayed wilting and maintained higher photosynthetic performance relative to controls, after exposure to soil dehydration. In each case, HvFC overexpression significantly upregulated the nuclear genes associated with detoxification of reactive oxygen species (ROS) upon drought stress. Overexpression of HvFCs, also suppressed photo-oxidative damage induced by the deregulated tetrapyrrole biosynthesis mutant tigrinad12. Previous studies suggest that only FC1 is implicated in stress defense responses, however, our study demonstrated that both FC1 and FC2 affect drought stress tolerance. As FC-derived free heme was proposed as a chloroplast-to-nuclear signal, heme could act as an important signal, stimulating drought responsive nuclear gene expression. This study also highlighted tetrapyrrole biosynthetic enzymes as potential targets for engineering improved crop performance, both in well-watered and water-limited environments

Altering Tetrapyrrole Biosynthesis by Overexpressing Ferrochelatases (Fc1 and Fc2) Improves Photosynthetic Efficiency in Transgenic Barley

Ferrochelatase (FC) is the terminal enzyme of heme biosynthesis. In photosynthetic organisms studied so far, there is evidence for two FC isoforms, which are encoded by two genes (FC1 and FC2). Previous studies suggest that these two genes are required for the production of two physiologically distinct heme pools with only FC2-derived heme involved in photosynthesis. We characterised two FCs in barley (Hordeum vulgare L.). The two HvFC isoforms share a common catalytic domain, but HvFC2 additionally contains a C-terminal chlorophyll a/b binding (CAB) domain. Both HvFCs are highly expressed in photosynthetic tissues, with HvFC1 transcripts also being abundant in non-photosynthetic tissues. To determine whether these isoforms differentially affect photosynthesis, transgenic barley ectopically overexpressing HvFC1 and HvFC2 were generated and evaluated for photosynthetic performance. In each case, transgenics exhibited improved photosynthetic rate (Asat), stomatal conductance (gs) and carboxylation efficiency (CE), showing that both FC1 and FC2 play important roles in photosynthesis. Our finding that modified FC expression can improve photosynthesis up to ~13% under controlled growth conditions now requires further research to determine if this can be translated to improved yield performance under field conditions

Insights into Long-Term Acclimation Strategies of Grapevines (<i>Vitis vinifera</i> L.) in Response to Multi-Decadal Cyclical Drought

Changing climatic conditions across Australia’s viticulture regions is placing increasing pressure on resources such as water and energy for irrigation. Therefore, there is a pressing need to identify superior drought tolerant grapevine clones by exploring the extensive genetic diversity of early European clones in old vineyards. Previously, in a field trial, we identified drought-tolerant (DT) dry-farmed Cabernet Sauvignon clones that had higher intrinsic water use efficiency (WUEi) under prolonged soil moisture deficiency compared to drought-sensitive (DS) clones. To investigate whether the field-grown clones have been primed and confer the drought-tolerant phenotypes to their subsequent vegetative progenies, we evaluated the drought responses of DT and DS progenies under two sequential drought events in a glasshouse alongside progenies of commercial clones. The DT clonal progenies exhibited improved gas exchange, photosynthetic performance and WUEi under recurrent drought events relative to DS clonal progenies. Concentration of a natural priming agent, γ-amino butyric acid (GABA), was significantly higher in DT progenies relative to other progenies under drought. Although DT and commercial clones displayed similar drought acclimation responses, their underlying hydraulic, stomatal and photosynthetic regulatory mechanisms were quite distinct. Our study provides fundamental insights into potential intergenerational priming mechanisms in grapevine

A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.)

Background: The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. Results: Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3-4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. Conclusion: The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes