Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase CO2 concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

Simultaneous and Independent Abaxial and Adaxial Gas Exchange Measurements.

Stomata can be distributed exclusively on the abaxial or adaxial leaf surface, but they are most commonly found on both leaf surfaces. Variations in stomatal arrangement, patterning, and the impact on photosynthesis can be measured using an infrared gas exchange system. However, when using standard gas exchange techniques, both surfaces are measured together and averaged to provide leaf-level values. Employing an innovative gas exchange apparatus with two infrared gas analyzers, separate gaseous flux from both leaf surfaces can be quantified simultaneously and independently. Here, we provide examples of typical measurements that can be performed using a "split chamber" gas exchange system.

Measuring Nonfoliar Photosynthesis.

Nonfoliar gas exchange provides a noninvasive way to measure photosynthesis and other gas exchange parameters, enabling the assessment of their potential contribution to yield. Photosynthesis in nonfoliar material has been gaining increasing attention due to its contribution to yield, especially under conditions when leaf photosynthesis is compromised. Here, we provide methods and approaches along with examples of measuring gas exchange in nonfoliar material using a specialized chamber.

Measurements of Carbon Assimilation in Aquatic Systems.

The recent development of an infrared gas analyzer capable of making carbon dioxide flux measurements from aquatic samples has enabled a new sphere of photosynthesis research. This study details key photosynthesis measurements on four aquatic and hydrophytic species, diverse in their morphology, physiology, and habitat. This guide specifies the methods and procedures needed to make reliable and accurate gas exchange measurements, with examples of data correction and presentation.

Tools for Measuring Photosynthesis at Different Scales.

Measurements of in vivo photosynthesis are powerful tools that probe the largest fluxes of carbon and energy in an illuminated leaf, but often the specific techniques used are so varied and specialized that it is difficult for researchers outside the field to select and perform the most useful assays for their research questions. The goal of this chapter is to provide a broad overview of the current tools available for the study of photosynthesis, both in vivo and in vitro, so as to provide a foundation for selecting appropriate techniques, many of which are presented in detail in subsequent chapters. This chapter will also organize current methods into a comparative framework and provide examples of how they have been applied to research questions of broad agronomical, ecological, or biological importance. This chapter closes with an argument that the future of in vivo measurements of photosynthesis lies in the ability to use multiple methods simultaneously and discusses the benefits of this approach to currently open physiological questions. This chapter, combined with the relevant methods chapters, could serve as a laboratory course in methods in photosynthesis research or as part of a more comprehensive laboratory course in general plant physiology methods.

Spectral Reflectance Measurements.

This chapter provides a methodology for evaluating plant health and leaf characteristics using spectral reflectance. It provides a step-by-step guide to using spectrometers for high-resolution point measurements of leaf spectral reflectance and multispectral imaging for capturing spatial data, emphasizing the importance of consistent measurement conditions. The chapter further explores the intricacies of multispectral imaging, including calibration, data collection, and image processing. Finally, this chapter delves into the application of various spectral indices for the quantification of key traits such as pigment content, the status of the xanthophyll cycle, water content, and how to identify spectral regions of interest for further research and development. Serving as a guide for researchers and practitioners in plant science, this chapter provides a straightforward framework for plant health assessment using spectral reflectance.

Using Infrared Thermography for High-Throughput Plant Phenotyping.

Infrared thermography offers a rapid, noninvasive method for measuring plant temperature, which provides a proxy for stomatal conductance and plant water status and can therefore be used as an index for plant stress. Thermal imaging can provide an efficient method for high-throughput screening of large numbers of plants. This chapter provides guidelines for using thermal imaging equipment and illustrative methodologies, coupled with essential considerations, to access plant physiological processes.

Imaging Spatial and Temporal Variation in Photosynthesis Using Chlorophyll Fluorescence.

Chlorophyll fluorescence imaging provides a noninvasive rapid screen to assess the physiological status of a number of leaves or plants simultaneously. Although there are no standard protocols for chlorophyll fluorescence imaging, here we provide an example of routines for some of the typical measurements.

Remote Sensing Techniques: Hyperspectral Imaging and Data Analysis.

Hyperspectral imaging is a remote sensing technique that enables remote, noninvasive measurement of plant traits. Here, we outline the procedures for camera setup, scanning, and calibration, along with the acquisition of black and white reference materials, which are the key steps in collecting hyperspectral imagery. We also discuss the development of predictive models such as partial least-squares regression, using both large and small datasets, which are used to predict plant traits from hyperspectral data. To ensure practical applicability, we provide code examples that allow readers to immediately implement these techniques in real-world scenarios. We introduce these topics to beginners in an accessible and understandable manner.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Excised leaves show limited and species-specific effects on photosynthetic parameters across crop functional types.

Photosynthesis is increasingly becoming a recognized target for crop improvement. Phenotyping photosynthesis-related traits on field-grown material is a key bottleneck to progress here due to logistical barriers and short measurement days. Many studies attempt to overcome these challenges by phenotyping excised leaf material in the laboratory. To date there are no demonstrated examples of the representative nature of photosynthesis measurements performed on excised leaves relative to attached leaves in crops. Here, we tested whether standardized leaf excision on the day prior to phenotyping affected a range of common photosynthesis-related traits across crop functional types using tomato (C3 dicot), barley (C3 monocot), and maize (C4 monocot). Potentially constraining aspects of leaf physiology that could be predicted to impair photosynthesis in excised leaves, namely leaf water potential and abscisic acid accumulation, were not different between attached and excised leaves. We also observed non-significant differences in spectral reflectance and chlorophyll fluorescence traits between the treatments across the three species. However, we did observe some significant differences between traits associated with gas exchange and photosynthetic capacity across all three species. This study represents a useful reference for those who perform measurements of this nature and the differences reported should be considered in associated experimental design and statistical analyses.

Engineering α-carboxysomes into plant chloroplasts to support autotrophic photosynthesis.

The growth in world population, climate change, and resource scarcity necessitate a sustainable increase in crop productivity. Photosynthesis in major crops is limited by the inefficiency of the key CO2-fixing enzyme Rubisco, owing to its low carboxylation rate and poor ability to discriminate between CO2 and O2. In cyanobacteria and proteobacteria, carboxysomes function as the central CO2-fixing organelles that elevate CO2 levels around encapsulated Rubisco to enhance carboxylation. There is growing interest in engineering carboxysomes into crop chloroplasts as a potential route for improving photosynthesis and crop yields. Here, we generate morphologically correct carboxysomes in tobacco chloroplasts by transforming nine carboxysome genetic components derived from a proteobacterium. The chloroplast-expressed carboxysomes display a structural and functional integrity comparable to native carboxysomes and support autotrophic growth and photosynthesis of the transplastomic plants at elevated CO2. Our study provides proof-of-concept for a route to engineering fully functional CO2-fixing modules and entire CO2-concentrating mechanisms into chloroplasts to improve crop photosynthesis and productivity.

Feeding the world: impacts of elevated [CO2] on nutrient content of greenhouse grown fruit crops and options for future yield gains.

Several long-term studies have provided strong support demonstrating that growing crops under elevated [CO2] can increase photosynthesis and result in an increase in yield, flavour and nutritional content (including but not limited to Vitamins C, E and pro-vitamin A). In the case of tomato, increases in yield by as much as 80% are observed when plants are cultivated at 1000 ppm [CO2], which is consistent with current commercial greenhouse production methods in the tomato fruit industry. These results provide a clear demonstration of the potential for elevating [CO2] for improving yield and quality in greenhouse crops. The major focus of this review is to bring together 50 years of observations evaluating the impact of elevated [CO2] on fruit yield and fruit nutritional quality. In the final section, we consider the need to engineer improvements to photosynthesis and nitrogen assimilation to allow plants to take greater advantage of elevated CO2 growth conditions.

The impact of growth at elevated [CO2] on stomatal anatomy and behavior differs between wheat species and cultivars.

The ability of plants to respond to changes in the environment is crucial to their survival and reproductive success. The impact of increasing the atmospheric CO2 concentration (a[CO2]), mediated by behavioral and developmental responses of stomata, on crop performance remains a concern under all climate change scenarios, with potential impacts on future food security. To identify possible beneficial traits that could be exploited for future breeding, phenotypic variation in morphological traits including stomatal size and density, as well as physiological responses and, critically, the effect of growth [CO2] on these traits, was assessed in six wheat relative accessions (including Aegilops tauschii, Triticum turgidum ssp. Dicoccoides, and T. turgidum ssp. dicoccon) and five elite bread wheat T. aestivum cultivars. Exploiting a range of different species and ploidy, we identified key differences in photosynthetic capacity between elite hexaploid wheat and wheat relatives. We also report differences in the speed of stomatal responses which were found to be faster in wheat relatives than in elite cultivars, a trait that could be useful for enhanced photosynthetic carbon gain and water use efficiency. Furthermore, these traits do not all appear to be influenced by elevated [CO2], and determining the underlying genetics will be critical for future breeding programmes.

Development of an accurate low cost NDVI imaging system for assessing plant health.

BACKGROUND: Spectral imaging is a key method for high throughput phenotyping that can be related to a large variety of biological parameters. The Normalised Difference Vegetation Index (NDVI), uses specific wavelengths to compare crop health and performance. Increasing the accessibility of spectral imaging systems through the development of small, low cost, and easy to use platforms will generalise its use for precision agriculture. We describe a method for using a dual camera system connected to a Raspberry Pi to produce NDVI imagery, referred to as NDVIpi. Spectral reference targets were used to calibrate images into values of reflectance, that are then used to calculated NDVI with improved accuracy compared with systems that use single references/standards. RESULTS: NDVIpi imagery showed strong performance against standard spectrometry, as an accurate measurement of leaf NDVI. The NDVIpi was also compared to a relatively more expensive commercial camera (Micasense RedEdge), with both cameras having a comparable performance in measuring NDVI. There were differences between the NDVI values of the NDVIpi and the RedEdge, which could be attributed to the measurement of different wavelengths for use in the NDVI calculation by each camera. Subsequently, the wavelengths used by the NDVIpi show greater sensitivity to changes in chlorophyll content than the RedEdge. CONCLUSION: We present a methodology for a Raspberry Pi based NDVI imaging system that utilizes low cost, off-the-shelf components, and a robust multi-reference calibration protocols that provides accurate NDVI measurements. When compared with a commercial system, comparable NDVI values were obtained, despite the fact that our system was a fraction of the cost. Our results also highlight the importance of the choice of red wavelengths in the calculation of NDVI, which resulted in differences in sensitivity between camera systems.

Longer epidermal cells underlie a quantitative source of variation in wheat flag leaf size.

The wheat flag leaf is the main contributor of photosynthetic assimilates to developing grains. Understanding how canopy architecture strategies affect source strength and yield will aid improved crop design. We used an eight-founder population to investigate the genetic architecture of flag leaf area, length, width and angle in European wheat. For the strongest genetic locus identified, we subsequently created a near-isogenic line (NIL) pair for more detailed investigation across seven test environments. Genetic control of traits investigated was highly polygenic, with colocalisation of replicated quantitative trait loci (QTL) for one or more traits identifying 24 loci. For QTL QFll.niab-5A.1 (FLL5A), development of a NIL pair found the FLL5A+ allele commonly conferred a c. 7% increase in flag and second leaf length and a more erect leaf angle, resulting in higher flag and/or second leaf area. Increased FLL5A-mediated flag leaf length was associated with: (1) longer pavement cells and (2) larger stomata at lower density, with a trend for decreased maximum stomatal conductance (Gsmax ) per unit leaf area. For FLL5A, cell size rather than number predominantly determined leaf length. The observed trade-offs between leaf size and stomatal morphology highlight the need for future studies to consider these traits at the whole-leaf level.

Photosynthesis - beyond the leaf.

Although leaves are considered the main site for photosynthesis, other green nonfoliar tissues can carry out considerable amounts of photosynthetic carbon assimilation. With photosynthesis, a potential target for improving crop productivity, physiology and contribution of nonfoliar tissues to overall plant carbon acquisition is gaining increasing attention. This review will provide an overview of nonfoliar photosynthesis, the role of stomata in these tissues and methodologies for quantification and the contribution to overall carbon gain.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.