Lauriebeth Leonelli

Improving photosynthesis and crop productivity by accelerating recovery from photoprotection

Faster light adaptation improves productivity Crop plants protect themselves from excess sunlight by dissipating some light energy as heat, readjusting their systems when shadier conditions prevail. But the photosynthetic systems do not adapt to fluctuating light conditions as rapidly as a cloud passes overhead, resulting in suboptimal photosynthetic efficiency. Kromdijk et al. sped up the adaptation process by accelerating interconversion of violaxanthin and zeaxanthin in the xanthophyll cycle and by increasing amounts of a photosystem II subunit. Tobacco plants tested with this system showed about 15% greater plant biomass production in natural field conditions. Science, this issue p. 857 Altering the regulation of light harvesting increases photosynthetic efficiency and biomass productivity in crop plants. Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. When sunlit leaves are shaded by clouds or other leaves, this protective dissipation continues for many minutes and reduces photosynthesis. Calculations have shown that this could cost field crops up to 20% of their potential yield. Here, we describe the bioengineering of an accelerated response to natural shading events in Nicotiana (tobacco), resulting in increased leaf carbon dioxide uptake and plant dry matter productivity by about 15% in fluctuating light. Because the photoprotective mechanism that has been altered is common to all flowering plants and crops, the findings provide proof of concept for a route to obtaining a sustainable increase in productivity for food crops and a much-needed yield jump.

Recognition of the Hyaloperonospora parasitica effector ATR13 triggers resistance against oomycete, bacterial, and viral pathogens

Phytopathogenic oomycetes cause some of the most devastating diseases affecting agricultural crops. Hyaloperonospora parasitica is a native oomycete pathogen of Arabidopsis and is related to other oomycete phytopathogens that include several species of Phytophthora, including the causal agent of potato late blight. Recently, four oomycete effector genes have been isolated, and several oomycete genomes have been sequenced. We have developed an efficient and genetically amenable system to test putative effector genes using the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. The H. parasitica effector protein ATR13 was delivered via P. syringae by fusing the ATR13 gene with the avrRpm1 type three secretion signal peptide, a bacterial sequence that allows transfer of proteins into the host cell through the bacterial type III secretion system. We also inserted ATR13 into the genome of the turnip mosaic virus, a single-stranded RNA virus. Our results show that delivery of ATR13 via the bacterial or viral pathogen triggers defense responses in plants containing the cognate resistance protein RPP13Nd, which restricts proliferation of both pathogens. Hence, recognition of ATR13 by RPP13 initiates defense responses that are effective against oomycete, bacterial and viral pathogens, pointing to a common defense mechanism. We have characterized regions of the RPP13Nd resistance protein that are essential for effector recognition and/or downstream signaling, using transient coexpression in Nicotiana benthamiana.

Structural elucidation and functional characterization of the Hyaloperonospora arabidopsidis effector protein ATR13

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity.

Global Analysis of Arabidopsis /Downy Mildew Interactions Reveals Prevalence of Incomplete Resistance and Rapid Evolution of Pathogen Recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogens overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.

An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants

Abstract Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae

Alga-derived lipids represent an attractive potential source of biofuels. However, lipid accumulation in algae is a stress response tightly coupled to growth arrest, thereby imposing a major limitation on productivity. To identify transcriptional regulators of lipid accumulation, we performed an integrative chromatin signature and transcriptomic analysis to decipher the regulation of lipid biosynthesis in the alga Chlamydomonas reinhardtii. Genome-wide histone modification profiling revealed remarkable differences in functional chromatin states between the algae and higher eukaryotes and uncovered regulatory components at the core of lipid accumulation pathways. We identified the transcription factor, PSR1, as a pivotal switch that triggers cytosolic lipid accumulation. Dissection of the PSR1-induced lipid profiles corroborates its role in coordinating multiple lipid-inducing stress responses. The comprehensive maps of functional chromatin signatures in a major clade of eukaryotic life and the discovery of a transcriptional regulator of algal lipid metabolism will facilitate targeted engineering strategies to mediate high lipid production in microalgae.

Regulation and Levels of the Thylakoid K+/H+ Antiporter KEA3 Shape the Dynamic Response of Photosynthesis in Fluctuating Light

Crop canopies create environments of highly fluctuating light intensities. In such environments, photoprotective mechanisms and their relaxation kinetics have been hypothesized to limit photosynthetic efficiency and therefore crop yield potential. Here, we show that overexpression of the Arabidopsis thylakoid K+/H+ antiporter KEA3 accelerates the relaxation of photoprotective energy-dependent quenching after transitions from high to low light in Arabidopsis and tobacco. This, in turn, enhances PSII quantum efficiency in both organisms, supporting that in wild-type plants, residual light energy quenching following a high to low light transition represents a limitation to photosynthetic efficiency in fluctuating light. This finding underscores the potential of accelerating quenching relaxation as a building block for improving photosynthetic efficiency in the field. Additionally, by overexpressing natural KEA3 variants with modification to the C-terminus, we show that KEA3 activity is regulated by a mechanism involving its lumen-localized C-terminus, which lowers KEA3 activity in high light. This regulatory mechanism fine-tunes the balance between photoprotective energy dissipation in high light and maximum quantum yield in low light, likely to be critical for efficient photosynthesis in fluctuating light conditions.

Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non-photochemical quenching and carotenoid biosynthesis

Summary Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non‐photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor‐intensive and time‐consuming generation of stable transgenic lines and mutant populations – approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ‐related genes from diverse organisms, we performed Agrobacterium tumefaciens‐mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.

Evolution of an atypical de-epoxidase for photoprotection in the green lineage.

Plants, algae and cyanobacteria need to regulate photosynthetic light harvesting in response to the constantly changing light environment. Rapid adjustments are required to maintain fitness because of a trade-off between efficient solar energy conversion and photoprotection. The xanthophyll cycle, in which the carotenoid pigment violaxanthin is reversibly converted into zeaxanthin, is ubiquitous among green algae and plants and is necessary for the regulation of light harvesting, protection from oxidative stress and adaptation to different light conditions1,2. Violaxanthin de-epoxidase (VDE) is the key enzyme responsible for zeaxanthin synthesis from violaxanthin under excess light. Here we show that the Chlorophycean VDE (CVDE) gene from the model green alga Chlamydomonas reinhardtii encodes an atypical VDE. This protein is not homologous to the VDE found in plants and is instead related to a lycopene cyclase from photosynthetic bacteria3. Unlike the plant-type VDE that is located in the thylakoid lumen, the Chlamydomonas CVDE protein is located on the stromal side of the thylakoid membrane. Phylogenetic analysis suggests that CVDE evolved from an ancient de-epoxidase that was present in the common ancestor of green algae and plants, providing evidence of unexpected diversity in photoprotection in the green lineage.

Major Transcriptome Reprogramming Underlies Floral Mimicry Induced by the Rust Fungus Puccinia monoica in Boechera stricta

Pucciniamonoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boechera stricta . Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. Although much ecological research has been performed on P . monoica -induced pseudoflowers, this system has yet to be investigated at the molecular or genomic level. To date, the molecular alterations underlying the development of pseudoflowers and the genes involved have not been described. To address this, we performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated. These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers. This work illustrates the complex perturbations induced by rust pathogens in their host plants, and provides a starting point for understanding the molecular mechanisms of pathogen-induced floral mimicry.