Mass screening of rice mutant populations at low CO2 for identification of lowered photorespiration and respiration rates.

Introduction: Identifying rice (Oryza sativa) germplasm with improved efficiency of primary metabolism is of utmost importance in order to increase yields. One such approach can be attained through screening genetically diverse populations under altered environmental conditions. Growth or treatment under low carbon dioxide (CO2) concentrations can be used as a means of revealing altered leaf photorespiration, respiration and other metabolic variants. Methods: We developed a pipeline for very high throughput treatment of gamma- and ethyl methanesulfonate- (EMS) induced mutant populations of IR64 rice seedlings at very low CO2 for 7 days. 1050 seedlings per batch at 5th leaf stage were exposed to 60 ppm CO2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll depletion using SPAD. Screening results showed a distinct difference between the mutants and the WTs. Results and discussion: The mean chlorophyll loss in WTs ranged from 65% to 11% respectively, whereas in the mutant lines chlorophyll loss ranged from 0 to 100%, suggesting considerable phenotypic variation. Rice mutants with a reduced chlorophyll reduction (<10%) were identified as 'Chlorophyll retention mutants' (CRMs) under low CO2 stress. In total, 1909 mutant lines (14,000 seedlings) were screened for chlorophyll content under 30 ppm CO2, with 26 lines selected for detailed screening. These 26 putative candidates were self-seeded to produce an M5 generation, used to determine the genetic control of the altered response to low CO2. Gas exchange of light and CO2 response revealed that there were significant variations among photosynthetic properties in two selected rice mutants. The CO2 compensation points in the absence of photorespiration and leaf respiration rates were lower than the WTs and anatomical analyses showed that CRM 29 had improved mesophyll cell area. We propose that this approach is useful for generating new material for breeding rice with improved primary metabolism.

Quantifying the influence of water deficit on root and shoot growth in wheat using X-ray Computed Tomography.

The potential increased frequency and severity of drought associated with environmental change represents a significant obstacle to efforts aimed at enhancing food security due to its impact on crop development, and ultimately, yield. Our understanding of the impact of drought on crop growth in terms of plant aerial tissues is much more advanced than knowledge of the below-ground impacts. We undertook an experiment using X-ray Computed Tomography that aimed to support measurements of infrared gas exchange from plant shoots with quantification of 3D root architecture traits and the associated soil structural characteristics. Winter wheat (cv. Zebedee) was assessed at two early growth stages (14 and 21 days) under four water treatments (100, 75, 50 and 25 % of a notional field capacity (FC) and across two soil types (sandy loam and clay loam)). Plants generally grew better (to a larger size) in sandy loam soil as opposed to clay loam soil, most likely due to the soil structure and the associated pore network. All plants grew poorly under extreme water stress and displayed optimal growth at 75 % of FC, as opposed to 100 %, as the latter was most likely too wet. The optimal matric potential for root and shoot growth, inferred from the water release curve for each soil type, was higher than that for photosynthesis, stomatal conductance and transpiration suggesting root and shoot growth was more affected by soil water content than photosynthesis-related characteristics under water deficit conditions. With incidences of drought likely to increase, identification of wheat cultivars that are more tolerant of these conditions is important. Studies that consider the impact of water stress on both plant shoots and roots, and the role of the soil pore system such as this offer considerable potential in supporting these efforts.

Nitrogen partitioning and remobilization in relation to leaf senescence, grain yield and protein concentration in Indian wheat cultivars.

Nitrogen (N) fertilizer represents a significant cost for the grower and may also have environmental impacts through nitrate leaching and N2O (a greenhouse gas) emissions associated with denitrification. The objectives of this study were to quantify the genetic variability in N partitioning and N remobilization in Indian spring wheat cultivars and identify traits for improved grain yield and grain protein content for application in breeding N-efficient cultivars. Twenty-eight bread wheat cultivars and two durum wheat cultivars were tested in field experiments in two years in Maharashtra, India. Growth analysis was conducted at anthesis and harvest to assess above-ground dry matter (DM) and dry matter and N partitioning. Flag-leaf photosynthesis rate (A max ), flag-leaf senescence rate and canopy normalized difference vegetation index (NDVI) were also assessed. Significant N × genotype level interaction was observed for grain yield and N-use efficiency. There was a positive linear association between post-anthesis flag-leaf A max and grain yield amongst the 30 genotypes under high N (HN) conditions. Flag-leaf A max was positively associated with N uptake at anthesis (AGNA). Under both HN and low N (LN) conditions, higher N uptake at anthesis was associated with delayed onset of flag-leaf senescence and higher grain yield. Under N limitation, there was a genetic negative correlation between grain yield and grain protein concentration. Deviation from this negative relationship (grain protein deviation or GPD) was related to genotypic differences in post-anthesis N uptake. It is concluded that N uptake at anthesis was an important determinant of flag-leaf photosynthesis rate and grain yield under high N conditions; while post-anthesis N uptake was an important determinant of GPD of wheat grown under low to moderate N conditions in India.

Author Correction: Rice plants overexpressing OsEPF1 show reduced stomatal density and increased root cortical aerenchyma formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rice plants overexpressing OsEPF1 show reduced stomatal density and increased root cortical aerenchyma formation.

Stomata are adjustable pores in the aerial epidermis of plants. The role of stomata is usually described in terms of the trade-off between CO2 uptake and water loss. Little consideration has been given to their interaction with below-ground development or diffusion of other gases. We overexpressed the rice EPIDERMAL PATTERNING FACTOR1 (OsEPF1) to produce rice plants with reduced stomatal densities, resulting in lowered leaf stomatal conductance and enhanced water use efficiency. Surprisingly, we found that root cortical aerenchyma (RCA) is formed constitutively in OsEPF1OE lines regardless of tissue age and position. Aerenchyma is tissue containing air-spaces that can develop in the plant root during stressful conditions, e.g. oxygen deficiency when it functions to increase O2 diffusion from shoot to root. The relationship with stomata is unknown. We conclude that RCA development and stomatal development are linked by two possible mechanisms: first that reduced stomatal conductance inhibits the diffusion of oxygen to the root, creating an oxygen deficit and stimulating the formation of RCA, second that an unknown EPF signalling pathway may be involved. Our observations have fundamental implications for the understanding of whole plant gas diffusion and root-to-shoot signalling events.

Genetic variation in N-use efficiency and associated traits in Indian wheat cultivars.

Nitrogen (N) fertilizer represents a significant cost for the grower and may also have environmental impacts through nitrate leaching and N2O (a greenhouse gas) emissions associated with denitrification. The objectives of this study were to quantify the genetic variability in N-use efficiency (NUE) in Indian spring wheat cultivars and identify traits for improved NUE for application in breeding. Twenty eight bread wheat cultivars and two durum wheat cultivars were tested in field experiments in two years in Maharashtra, India. Detailed growth analysis was conducted at anthesis and harvest including dry matter (DM) and N partitioning. Senescence of the flag leaf was assessed from a visual score every 3-4 days from anthesis to complete flag-leaf senescence and fitted against thermal time to estimate the onset and end of post-anthesis senescence. Grain yield (GY) was reduced under low N (LN) by an average of 1.46 t ha-1 (-28%). Significant N × genotype level interaction was observed for grain yield and NUE. Above-ground N uptake at harvest was reduced from 162 kg N ha-1 under high N (HN) to 85 kg N ha-1 under low N (LN) conditions, while N-utilization efficiency (grain DM yield per unit crop N uptake at harvest; NUtE) increased from 32.7 to 44.6 kg DM kg-1 N. Genetic variation in GY under LN related mainly to variation in N uptake at harvest rather than NUtE; and the N × genotype effect for GY was mainly explained by the interaction for N uptake at harvest. Averaging across years, the linear regression of onset of flag-leaf senescence on GY amongst cultivars was significant under both HN (R2 0.16. p < 0.05) and LN (R2 0.21, p < 0.05) conditions. Onset of flag-leaf senescence was positively associated with N uptake at anthesis under HN (R2 0.34, p < 0.001) and LN (R2 0.22, p < 0.01) conditions. Flag-leaf senescence timing was not associated with post-anthesis N uptake. It is concluded that increased N accumulation at anthesis was correlated with flag-leaf senescence timing and that N accumulation at anthesis is an important trait for enhancing grain yield and NUE of wheat grown under low to moderate N supply in India.

Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications.

Chlorophyll fluorescence is a non-invasive measurement of photosystem II (PSII) activity and is a commonly used technique in plant physiology. The sensitivity of PSII activity to abiotic and biotic factors has made this a key technique not only for understanding the photosynthetic mechanisms but also as a broader indicator of how plants respond to environmental change. This, along with low cost and ease of collecting data, has resulted in the appearance of a large array of instrument types for measurement and calculated parameters which can be bewildering for the new user. Moreover, its accessibility can lead to misuse and misinterpretation when the underlying photosynthetic processes are not fully appreciated. This review is timely because it sits at a point of renewed interest in chlorophyll fluorescence where fast measurements of photosynthetic performance are now required for crop improvement purposes. Here we help the researcher make choices in terms of protocols using the equipment and expertise available, especially for field measurements. We start with a basic overview of the principles of fluorescence analysis and provide advice on best practice for taking pulse amplitude-modulated measurements. We also discuss a number of emerging techniques for contemporary crop and ecology research, where we see continual development and application of analytical techniques to meet the new challenges that have arisen in recent years. We end the review by briefly discussing the emerging area of monitoring fluorescence, chlorophyll fluorescence imaging, field phenotyping, and remote sensing of crops for yield and biomass enhancement.

Variation in vein density and mesophyll cell architecture in a rice deletion mutant population.

There is a need to develop rice plants with improved photosynthetic capacity and efficiency in order to enhance potential grain yield. Alterations in internal leaf morphology may be needed to underpin some of these improvements. One target is the production of a 'Kranz-like' anatomy, commonly considered to be required to achieve the desired levels of photosynthesis seen in C(4) crops. Kranz anatomy typically has two or three mesophyll cells interspersing adjacent veins. As a first step to determining the potential for such anatomical modifications in rice leaves, a population of rice deletion mutants was analysed for alterations in vein patterning and mesophyll cells in the interveinal regions. Significant variation is demonstrated in vein arrangement and the sequential distribution of major and minor veins across the leaf width, although there is a significant correlation between the total number of veins present and the width of the leaf. Thus the potential is demonstrated for modifying rice leaf structure. Six distinct rice mutant lines, termed altered leaf morphology (alm) mutants, were analysed for the architecture of their interveinal mesophyll cell arrangement. It is shown that in these mutant lines, the distance between adjacent minor veins and adjacent minor and major veins is essentially determined by the size of the interveinal mesophyll cells rather than changes in mesophyll cell number across this region, and hence interveinal distance changes as a result of cell expansion rather than cell division. This observation will be important when developing screens for traits relevant for the introduction of Kranz anatomy into rice.

Agriculture and the new challenges for photosynthesis research.

A rising human population and changing patterns of land use mean that world food production rates will need to be increased by at least 50% by 2050, a massive rise in harvestable yield per hectare of the major crops such as rice (Oryza sativa) and wheat (Triticum aestivum). Combinations of breeding for improved morphology-related traits such as harvest index and increased inputs of water and fertilizer, which have sustained yield increases since the 1960s, will be neither sufficient nor sustainable. An important limiting factor will be the capacity to produce sufficient biomass during favourable growing periods. Here we analyse this problem in the context of increasing the efficiency of conversion of solar energy into biomass, that is, leaf and canopy photosynthesis. Focussing on crops carrying out C3 photosynthesis, we analyse the evidence for 'losses' in the process of conversion of solar energy into crop biomass and we explore novel mechanisms of improving biomass production rates, which have arisen from recent research into the fundamental primary processes of photosynthesis and carbohydrate metabolism. We show that there are several lines of evidence that these processes are not fully optimized for maximum yield. We put forward the hypothesis that the chloroplast itself should be given greater prominence as a sensor, processor and integrator of highly variable environmental signals to allow a more efficient transduction of energy supply into biomass production.

Trends in leaf photosynthesis in historical rice varieties developed in the Philippines since 1966.

Crop improvement in terms of yield is rarely linked to leaf photosynthesis. However, in certain crop plants such as rice, it is predicted that an increase in photosynthetic rate will be required to support future grain yield potential. In order to understand the relationships between yield improvement and leaf photosynthesis, controlled environment conditions were used to grow 10 varieties which were released from the International Rice Research Institute (IRRI) between 1966 and 1995 and one newly developed line. Two growth light intensities were used: high light (1500 micromol m(-2) s(-1)) and low light (300 micromol m(-2) s(-1)). Gas exchange, leaf protein, chlorophyll, and leaf morphology were measured in the ninth leaf on the main stem. A high level of variation was observed among high light-grown plants for light-saturated photosynthetic rate per unit leaf area (P(max)), stomatal conductance (g), content of ribulose bisphosphate carboxylase-oxygenase (Rubisco), and total leaf protein content. Notably, between 1966 and 1980 there was a decline in P(max), g, leaf protein, chlorophyll, and Rubisco content. Values recovered in those varieties released after 1980. This striking trend coincides with a previous published observation that grain yield in IRRI varieties released prior to 1980 correlated with harvest index whereas that for those released after 1980 correlated with biomass. P(max) showed significant correlations with both g and Rubisco content. Large differences were observed between high light- and low light-grown plants (photoacclimation). The photoacclimation 'range' for P(max) correlated with P(max) in high light-grown plants. It is concluded that (i) leaf photosynthesis may be systematically affected by breeding strategy; (ii) P(max) is a useful target for yield improvements where yield is limited by biomass production rather than partitioning; and (iii) the capacity for photoacclimation is related to high P(max) values.

Acclimation of photosynthesis to high irradiance in rice: gene expression and interactions with leaf development.

Rice (Oryza sativa L.) has been used to study the long-term responses of photosynthesis to high irradiance focusing on the composition of the photosynthetic apparatus and leaf morphology. Typical sun/shade differences in chloroplast composition are seen in the fifth leaf following growth in high irradiance compared with low irradiance (1000 and 200 micromol m(-2) s(-1), respectively): higher light-saturated rates of photosynthesis (P(max)), higher amounts of Rubisco protein, and a lower chlorophyll a:b ratio. In addition, leaves were thicker under high light compared with low light. However, responses appear more complex when leaf developmental stage is considered. Using a system of transferring plants from low to high light in the laboratory responses that occur before and after full leaf extension have been studied. Acclimation of photosynthesis is limited by leaf age: the transfer to high light, post-leaf extension, is characterized by alterations in chlorophyll a:b but not in Rubisco protein, which may be limited by leaf morphology. Microarray analysis of gene expression was carried out on plants that were transferred to high light post-leaf extension. A down-regulation of light-harvesting genes was seen. No change in the expression level of Rubisco genes was observed. Up-regulation of genes involved in photoprotection was observed. It was also shown that high-light leaf morphology is established prior to formation of the zone of cellular elongation and division. The endogenous and environmental factors which establish the characteristics of high light acclimation may be important for attaining high rates of assimilation in leaves and crop canopies, and the fifth leaf in rice provides a convenient model system for the determination of the mechanisms involved.

Increasing rice photosynthesis by manipulation of the acclimation and adaptation to light.

There are three important considerations in assessing the interaction of crop plants with light: (a) how does the plant respond to the light environment both in the short-term (regulation) and in the long-term (acclimation), (b) under what conditions are these responses inadequate, leading to photoinhibition, and (c) are the responses optimally adapted for maximum agricultural yield? Despite a wealth of knowledge about these processes in model plant species, it is impossible to predict how significant they are in influencing the yield of rice. Therefore, in collaboration with IRRI, we have undertaken a study of photoinhibition and photoacclimation of rice under field conditions. The results of this study are presented, along with an assessment of the implications for improvement of rice yield.

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves.

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.