Mining for allelic gold: finding genetic variation in photosynthetic traits in crops and wild relatives.

Improvement of photosynthetic traits in crops to increase yield potential and crop resilience has recently become a major breeding target. Synthetic biology and genetic technologies offer unparalleled opportunities to create new genetics for photosynthetic traits driven by existing fundamental knowledge. However, large 'gene bank' collections of germplasm comprising historical collections of crop species and their relatives offer a wealth of opportunities to find novel allelic variation in the key steps of photosynthesis, to identify new mechanisms and to accelerate genetic progress in crop breeding programmes. Here we explore the available genetic resources in food and fibre crops, strategies to selectively target allelic variation in genes underpinning key photosynthetic processes, and deployment of this variation via gene editing in modern elite material.

Leaf N content regulates the speed of photosynthetic induction under fluctuating light among canola genotypes (Brassica napus L.).

Nitrogen is an essential element for plant growth, and the relationship between leaf N content and photosynthesis has been widely studied in different species under steady-state light. However, under natural conditions, the light intensity at the leaf level is always changing, inherently heterogeneous in time and space. Therefore, the effect of leaf N content on photosynthesis under dynamic light conditions needs further study. At present, the effects of leaf N content on leaf non-steady-state photosynthesis have not been reported in canola (Brassica napus L.). To clarify the relationship between leaf N content and the speed of the response leaf gas exchange to variations in light intensity, eight genotypes of canola varying in leaf N content were used to study the temporal response of gas exchange to a step increase in irradiance. We found there were significant differences in non-steady-state photosynthesis, physiological characteristics, and anatomical traits across genotypes (the maximum amplitude was about fivefold), despite the lack of contrast under normal, steady-state photosynthesis. In addition, initial stomatal conductance to water vapor in the darkness and leaf N content per leaf area were negatively correlated with the time required to achieve 50% and 100% of the maximum photosynthetic rate. Contrarily, the time required to reach 50% of the maximum stomatal conductance was positively correlated with the time required to achieve 90% of the maximum photosynthetic rate across genotypes. It is concluded that the genotypes of canola with higher N content per leaf area show a faster induction of photosynthesis to fluctuating light conditions.

The PAP/SAL1 retrograde signaling pathway is involved in iron homeostasis.

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.

Photons to food: genetic improvement of cereal crop photosynthesis.

Photosynthesis has become a major trait of interest for cereal yield improvement as breeders appear to have reached the theoretical genetic limit for harvest index, the mass of grain as a proportion of crop biomass. Yield improvements afforded by the adoption of green revolution dwarfing genes to wheat and rice are becoming exhausted, and improvements in biomass and radiation use efficiency are now sought in these crops. Exploring genetic diversity in photosynthesis is now possible using high-throughput techniques, and low-cost genotyping facilitates discovery of the genetic architecture underlying this variation. Photosynthetic traits have been shown to be highly heritable, and significant variation is present for these traits in available germplasm. This offers hope that breeding for improved photosynthesis and radiation use efficiency in cereal crops is tractable and a useful shorter term adjunct to genetic and genome engineering to boost yield potential.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.

Wheat drought tolerance in the field is predicted by amino acid responses to glasshouse-imposed drought.

Water limits crop productivity, so selecting for a minimal yield gap in drier environments is critical to mitigate against climate change and land-use pressure. We investigated the responses of relative water content (RWC), stomatal conductance, chlorophyll content, and metabolites in flag leaves of commercial wheat (Triticum aestivum L.) cultivars to three drought treatments in the glasshouse and in field environments. We observed strong genetic associations between glasshouse-based RWC, metabolites, and yield gap-based drought tolerance (YDT; the ratio of yield in water-limited versus well-watered conditions) across 18 field environments spanning sites and seasons. Critically, RWC response to glasshouse drought was strongly associated with both YDT (r2=0.85, P<8E-6) and RWC under field drought (r2=0.77, P<0.05). Moreover, multiple regression analyses revealed that 98% of genetic YDT variance was explained by drought responses of four metabolites: serine, asparagine, methionine, and lysine (R2=0.98; P<0.01). Fitted coefficients suggested that, for given levels of serine and asparagine, stronger methionine and lysine accumulation was associated with higher YDT. Collectively, our results demonstrate that high-throughput, targeted metabolic phenotyping of glasshouse-grown plants may be an effective tool for selection of wheat cultivars with high field-derived YDT.

Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.

Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light.

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a change in the reduced/oxidized (redox) state of photosynthetic electron transport components in chloroplasts. However, how this generates a signal that is relayed to changes in nuclear gene expression is not well understood. We modified redox state in the reference plant, Arabidopsis thaliana, using either excess light or low light plus the herbicide DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), a well-known inhibitor of photosynthetic electron transport. Modification of redox state caused a change in expression of a common set of about 750 genes, many of which are known stress-responsive genes. Among the most highly enriched promoter elements in the induced gene set were heat-shock elements (HSEs), known motifs that change gene expression in response to high temperature in many systems. We show that HSEs from the promoter of the ASCORBATE PEROXIDASE 2 (APX2) gene were necessary and sufficient for APX2 expression in conditions of excess light, or under low light plus the herbicide. We tested APX2 expression phenotypes in overexpression and loss-of-function mutants of 15 Arabidopsis A-type heat-shock transcription factors (HSFs), and identified HSFA1D, HSFA2, and HSFA3 as key factors regulating APX2 expression in diverse stress conditions. Excess light regulates both the subcellular location of HSFA1D and its biochemical properties, making it a key early component of the excess light stress network of plants.

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.

Using tunable diode laser spectroscopy to measure carbon isotope discrimination and mesophyll conductance to CO2 diffusion dynamically at different CO2 concentrations.

In C3 leaves, the mesophyll conductance to CO2 diffusion, g(m) , determines the drawdown in CO2 concentration from intercellular airspace to the chloroplast stroma. Both g(m) and stomatal conductance limit photosynthetic rate and vary in response to the environment. We investigated the response of g(m) to changes in CO2 in two Arabidopsis genotypes (including a mutant with open stomata, ost1), tobacco and wheat. We combined measurements of gas exchange with carbon isotope discrimination using tunable diode laser absorption spectroscopy with a CO2 calibration system specially designed for a range of CO2 and O2 concentrations. CO2 was initially increased from 200 to 1000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm, or the sequence was reversed. In 2% O2 a step increase from 200 to 1000 ppm significantly decreased g(m) by 26-40% in all three species, whereas following a step decrease from 1000 to 200 ppm, the 26-38% increase in g(m) was not statistically significant. The response of g(m) to CO2 was less in 21% O2. Comparing wild type against the ost1 revealed that mesophyll and stomatal conductance varied independently in response to CO2. We discuss the effects of isotope fractionation factors on estimating g(m) .