Chloroplasts act as environmental sensors, enabling rapid plant stress responses through operational retrograde signaling. While these signals operate on minutes-to-hours' time scales, their cumulative impact and function across a plant's lifecycle and in field conditions remains unknown. We investigated if retrograde signaling's transient changes to gene expression have cumulative effects by generating wheat mutants with primed SAL1-PAP retrograde pathway responsiveness. After confirming changes in photosynthesis and drought resilience under controlled conditions, we conducted 15 field trials across South-Eastern Australia, spanning diverse environments with varying temperatures, rainfall, and light conditions, analysing physiological responses, yield, biomass, and water productivity. We found gene locus-specific effects on biomass, yield, and water productivity. Multi-environment analysis showed the SAL-4A locus was more strongly associated with improved performance, with the converse for SAL-5D. Significantly, modulation of specific SAL subtypes enhanced photosynthetic efficiency and stress resilience while improving average yields by 4 and 8% respectively for 4A loci across 15 field sites, challenging the traditional yield-resilience trade-off paradigm. This study reveals retrograde signals operate in field environments, integrating environmental information across a plant's lifecycle to improve yield, water productivity and dynamic acclimation to diverse growing seasons, and highlight the importance of multi-environment field validation for crop modifications. ### Competing Interest Statement The authors have declared no competing interest.

Metabolically engineered high-leaf oil plants have been developed to meet the increasing demand for plant oils. Oil production of these plants under controlled conditions is promising; however, their performance under field-like conditions with abiotic stresses remains uncertain. In this study, wild-type (WT) and high-leaf oil (HLO) transgenic tobacco (Nicotiana tabacum) plants were exposed to moderate and sustained water stress to mimic field conditions. The effects of water stress on biomass and lipid accumulation were investigated at the physiological, biochemical, and transcriptional levels. The presence of transgenes increased leaf triacylglycerol (TAG) levels in HLO plants by upregulating endogenous genes involved in lipid biosynthesis at the expense of biomass reduction, altered leaf lipid content and profile, and a decrease in unsaturation levels of membrane lipids compared to WT plants. Moreover, the biomass penalty in HLO plants could reduce canopy transpiration, contributing to their better performance under water-limited environments. Furthermore, WT and HLO plants exhibited enhanced TAG accumulation under water stress but via different mechanisms. In WT plants, water stress induced lipid remodeling, upregulated genes encoding phosphatidic acid phosphatase (PAP), diacylglycerol o-acyltransferase (DGAT2), and lipid droplet-associated proteins (LDAP1), but downregulated genes encoding Gly-Asp-Ser-Leu (GDSL) lipases. In contrast, HLO plants showed increased TAG accumulation primarily through upregulation of OLEOSINS and downregulation of GDSLs under water stress. In conclusion, moderate water stress promoted oil production in HLO plants, demonstrating the robustness of HLO technology for sustainable oil production in the field under water deficit conditions which may be more prevalent in the future due to climate change.

Many C4 plants are used as food and fodder crops and often display improved resource use efficiency compared to C3 plants. However, the response of C4 plants to future extreme conditions such as heatwaves is less understood. Here, Setaria viridis, an emerging C4 model grass, was grown under long-term high-temperature stress for 2 wk (42 degrees C, compared to 28 degrees C). This resulted in stunted growth, but surprisingly had little impact on leaf thickness, leaf area-based photosynthetic rates, and bundle sheath leakiness. Dark respiration rates increased, and there were major alterations in carbon and nitrogen metabolism in the heat-stressed plants. Abscisic acid and indole-3-acetic acid-amino acid conjugates accumulated in the heat-stressed plants, consistent with transcriptional changes. Leaf transcriptomics, proteomics, and metabolomics analyses were carried out and mapped onto the metabolic pathways of photosynthesis, respiration, carbon/nitrogen metabolism, and phytohormone biosynthesis and signaling. An in-depth analysis of correlations between transcripts and their corresponding proteins revealed strong differences between groups in the strengths and signs of correlations. Overall, many stress signaling pathways were upregulated, consistent with multiple signals leading to reduced plant growth. A systems-based model of the plant response to long-term heat stress is presented based on the oxidative stress, phytohormone, and sugar signaling pathways. Long-term heat stress does not affect photosynthesis in the C4 grass Setaria viridis owing to an effective acclimation response of carbon assimilation, although it inhibits plant growth.

Chloroplast-to-nucleus retrograde signalling enables rapid stress responses in plants, but whether these signals accumulate to affect crop performance across entire growing seasons under field conditions remains unknown. We generated wheat mutants with targeted deletions in specific SAL gene copies from two distinct homeologous groups (TaSAL1 and TaSAL2), creating lines with enhanced stress signal responsiveness. We tested these lines across 15 field trials spanning diverse Australian environments with varying temperatures, rainfall, and irrigation regimes, measuring physiological responses, yield, biomass, and water productivity. Lines with TaSAL2 gene deletions showed 4-8% yield improvements with enhanced water productivity, while TaSAL1 deletions reduced yields. The TaSAL2 mutants maintained superior photosynthetic function under drought stress, showed improved relative water content, and demonstrated enhanced yield stability across environments. Canopy temperature measurements revealed dynamic stomatal regulation, with increased closure during midday stress periods but normal aperture under benign conditions. Significantly, specific SAL modifications enhanced photosynthetic efficiency and stress resilience without traditional yield penalties. Targeted modification of specific SAL homeologous groups can simultaneously improve both yield and stress tolerance in wheat. This demonstrates that retrograde signalling integrates environmental information across the plant lifecycle and highlights the importance of locus-specific targeting and multienvironment field validation for crop modifications.

BACKGROUND:The need for rapid in-field measurement of key traits contributing to yield over many thousands of genotypes is a major roadblock in crop breeding. Recently, leaf hyperspectral reflectance data has been used to train machine learning models using partial least squares regression (PLSR) to rapidly predict genetic variation in photosynthetic and leaf traits across wheat populations, among other species. However, the application of published PLSR spectral models is limited by a fixed spectral wavelength range as input and the requirement of separate custom-built models for each trait and wavelength range. In addition, the use of reflectance spectra from the short-wave infrared region requires expensive multiple detector spectrometers. The ability to train a model that can accommodate input from different spectral ranges would potentially make such models extensible to more affordable sensors. Here we compare the accuracy of prediction of PLSR with various deep learning approaches and an ensemble model, each trained and tested using previously published data sets. RESULTS:We demonstrate that the accuracy of PLSR to predict photosynthetic and related leaf traits in wheat can be improved with deep learning-based and ensemble models without overfitting. Additionally, these models can be flexibly applied across spectral ranges without significantly compromising accuracy. CONCLUSION:The method reported provides an improved prediction of wheat leaf and photosynthetic traits from leaf hyperspectral reflectance and do not require a full range, high cost leaf spectrometer. We provide a web service for deploying these algorithms to predict physiological traits in wheat from a variety of spectral data sets, with important implications for wheat yield prediction and crop breeding.

Drought is a major threat to food security. Water loss through stomata is an inevitable consequence of CO2 uptake, and water deficit inhibits plant growth, making it challenging to develop drought-resistant strategies without compromising yield. Here, we generated tobacco plants expressing a maize NADP-dependent malate decarboxylating enzyme in stomata and vascular cells (ME plants), which show higher seed yield and faster maturation compared to wild-type (WT) plants under normal irrigation and after drought. While WT plants die after 45 days of drought, ME plants survive without any adverse effects on seed production. In addition, ME plants exhibit improved photosynthetic efficiency despite reduced stomatal conductance and changes in stem morphology, which are likely related to their ability to withstand drought. We propose that enhanced C4-like biochemistry in cells surrounding the vascular system and increased sugar export likely compensated for the reduced stomatal conductance in ME plants. The study demonstrates that cell-targeted metabolic modifications can avoid pleiotropic effects and facilitate the stacking of beneficial traits to improve crop design. Significance Statement Drought is one of the biggest threats to global food security, and its impact on crop yield is expected to worsen due to climate change. Traditionally, drought resistance has often come at the expense of yield, creating a negative trade-off. However, we present here a promising solution to this challenge. We have developed a novel approach that successfully uncouples the negative balance between drought resistance and yield. By introducing a maize enzyme into specific tobacco cells, we have created drought-resistant plants with faster growth and higher seed yield. Most importantly, after prolonged drought, while the wild type dies, the modified plants maintain their high yield. This technology paves the way for greater food security and resilience to climate change. ### Competing Interest Statement The authors have declared no competing interest.

A substantial number of C 4 plants are utilized as food and fodder crops and often display improved resource use efficiency compared to C 3 counterparts. However, their response to future extreme climates such as heatwaves is less understood. Setaria viridis , an emerging C 4 model grass closely related to important C4 crops, was grown under high temperature for two weeks (42°C as compared to 28°C). High temperature resulted in stunted growth, but surprisingly had little impact on leaf area based photosynthetic rates. Rates of dark respiration significantly increased and there were major alterations in carbon and nitrogen metabolism in the heat-stressed plants, including reduced starch levels, accumulation of soluble sugars and an increase in leaf nitrogen content. Measurements of major phytohormones revealed a dramatic increase in abscisic acid in the heat-stressed plants. Leaf transcriptomics, proteomics and metabolomics analyses were carried out and mapped onto metabolic pathways of photosynthesis, respiration, carbon/nitrogen metabolism and hormone synthesis and signaling. Overall, upregulation of a number of stress-signaling pathways was observed, consistent with multiple potent signals leading to reduced plant growth. A systems model of plant response is presented based on oxidative stress, hormone and sugar signaling pathways.

With population growth and a shrinking rural workforce, agricultural technologies have become increasingly important. Above-ground biomass (AGB) is a key trait relevant to breeding, agronomy and crop physiology field experiments. However, measuring the biomass of a cereal plot requires cutting, drying and weighing processes, which are laborious, expensive and destructive tasks. This paper proposes a non-destructive and high-throughput method to predict biomass from field samples based on Light Detection and Ranging (LiDAR). Unlike previous methods that are based on the density of a point cloud or plant height, our biomass prediction network (BioNet) additionally considers plant structure. Our BioNet contains three modules: 1) a completion module to predict missing points due to canopy occlusion; 2) a regularization module to regularize the neural representation of the whole plot; and 3) a projection module to learn the salient structures from a bird’s eye view of the point cloud. An attention-based fusion block is used to achieve final biomass predictions. In addition, the complete dataset, including hand-measured biomass and LiDAR data, is made available to the community. Experiments show that our BioNet achieves ≈ 33% improvement over current state-of-the-art methods.

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.

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.

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.

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 3ʹ-phosphoadenisine-5ʹ-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.

The wheat Sr2 locus confers partial resistance to four biotrophic pathogens: wheat stem rust (Puccinia graminis f. sp. tritici), leaf rust (P. triticina), stripe rust (P. striiformis f. sp. tritici), and powdery mildew (Blumeria graminis f. sp. tritici). In addition, Sr2 is linked with a brown coloration of ears and stems, termed pseudo-black chaff (PBC). PBC, initially believed to be elicited by stem rust infection, was subsequently recognized to occur in the absence of pathogen infection. The current study demonstrates that the resistance response to stem rust is associated with the death of photosynthetic cells around rust infection sites in the inoculated leaf sheath. Similarly, Sr2-dependent resistance to powdery mildew was associated with the death of leaf mesophyll cells around mildew infection sites. We demonstrate that PBC occurring in the absence of pathogen inoculation also corresponds with death and the collapse of photosynthetic cells in the affected parts of stems and ears. In addition, Sr2-dependent necrosis was inducible in leaves by application of petroleum jelly or by heat treatments. Thus, Sr2 was found to be associated with cell death, which could be triggered by either biotic or abiotic stresses. Our results suggest a role for the Sr2 locus in controlling cell death in response to stress.

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.

Biocatalytic transformation of carbamate formed readily from CO2 and NH3 provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO2 and NH3. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in E. coli with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by E. coli cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric ATP (>300 turnovers) as well as its in situ reaction with ornithine to give citrulline in high yield (>95%) and g L-1 h(-1) titers. The system is active over a wide range of NH3 concentrations (2.5 mM - 2 M), and >90% conversions of NH3 may be reached within 1.5 h. Aqueous NH3 used to sequester CO2 gas (10% v/v) may be directly used as the biocatalyst feedstock. In preliminary studies, citrulline is found to be an effective organic nitrogen fertilizer of the wheat grass Brachypodium distachyon. Therefore, lysates described here constitute a cost-effective biocatalytic platform for one-pot production of a promising organic nitrogen fertilizer, under mild reaction conditions, from environmental pollutants as feedstock.

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3'-phosphoadenosine-5'-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.

Abstract Homeostasis of metabolism and regulation of stress‐signaling pathways are important for plant growth. The metabolite 3′‐phosphoadenosine‐5′‐phosphate (PAP) plays dual roles as a chloroplast retrograde signal during drought and high light stress, as well as a toxic by‐product of secondary sulfur metabolism, and thus, its levels are regulated by the chloroplastic phosphatase, SAL1. Constitutive PAP accumulation in sal1 mutants improves drought tolerance but can impair growth and alter rosette morphology. Therefore, it is of interest to derive strategies to enable controlled and targeted PAP manipulation that could enhance drought tolerance while minimizing the negative effects on plant growth. We systematically tested the potential and efficiency of multiple established transgenic manipulation tools in altering PAP levels in Arabidopsis. Dexamethasone (dex)‐inducible silencing of SAL1 via hpRNAi [pOpOff:SAL1hpRNAi] yielded reduction in SAL1 transcript and protein levels, yet failed to significantly induce PAP accumulation. Surprisingly, this was not due to insufficient silencing of the inducible system, as constitutive silencing using a strong promoter to drive hpRNAi and amiRNA targeting the SAL1 transcript also failed to increase PAP content or induce a sal1‐like plant morphology despite significantly reducing the SAL1 transcript levels. In contrast, using dex‐inducible expression of SAL1 cDNA to complement an Arabidopsis sal1 mutant successfully modulated PAP levels and restored rosette growth in a dosage‐dependent manner. Results from this inducible complementation system indicate that plants with intermediate PAP levels could have improved rosette growth without compromising its drought tolerance. Additionally, preliminary evidence suggests that SAL1 cDNA driven by promoters of genes expressed specifically during early developmental stages such as ABA‐Insensitive 3 (ABI3) could be another potential strategy for studying and optimizing PAP levels and drought tolerance while alleviating the negative impact of PAP on plant growth in sal1. Thus, we have identified ways that can allow future dissection into multiple aspects of stress and developmental regulation mediated by this chloroplast signal.

We examined the function of OsALMT4 in rice (Oryza sativa L.) which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ABA, IAA, and salicylic acid. Malate efflux from the transgenic plants over-expressing OsALMT4 was inhibited by niflumate and salicylic acid. Growth of transgenic lines with either increased OsALMT4 expression or reduced expression was measured in different environments. Light intensity caused significant differences in growth between the transgenic lines and controls. When day-time light was reduced from 700 to 300 μmol m-2s-1 independent transgenic lines with either increased or decreased OsALMT4 expression accumulated less biomass compared to their null controls. This response was not associated with differences in photosynthetic capacity, stomatal conductance or sugar concentrations in tissues. We propose that by disrupting malate fluxes across the plasma membrane carbon partitioning and perhaps signaling are affected which compromises growth under low light. We conclude that OsALMT4 is expressed widely in rice and facilitates malate efflux from different cell types. Altering OsALMT4 expression compromises growth in low-light environments.

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.