The importance of species-specific and temperature-sensitive parameterisation of A/Ci models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO2 assimilation to variable intercellular CO2 concentrations (A/Ci response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, Vcmax) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; JPAR). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO2 (gm) derived from tobacco that may be inapplicable across species. To study the significance of using such parameters for other species, here we measured the temperature responses of key in vitro Rubisco catalytic properties and gm in cotton (Gossypium hirsutum cv. Sicot 71) and derived Vcmax and J2000 (JPAR at 2000 µmol m-2 s-1 PAR) from cotton A/Ci curves incrementally measured at 15°C-40°C using cotton and other species-specific sets of input parameters with our new automated fitting R package 'OptiFitACi'. Notably, parameterisation by a set of tobacco parameters produced unrealistic J2000:Vcmax ratio of <1 at 25°C, two- to three-fold higher estimates of Vcmax above 15°C, up to 2.3-fold higher estimates of J2000 and more variable estimates of Vcmax and J2000, for our cotton data compared to model parameterisation with cotton-derived values. We determined that errors arise when using a gm,25 of 2.3 mol m-2 s-1 MPa-1 or less and Rubisco CO2-affinities in 21% O2 (KC 21%O2) at 25°C outside the range of 46-63 Pa to model A/Ci responses in cotton. We show how the A/Ci modelling capabilities of 'OptiFitACi' serves as a robust, user-friendly, and flexible extension of 'plantecophys' by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to reduce variability when modelling Vcmax and J2000.

A standardised approach for determining heat tolerance in cotton using triphenyl tetrazolium chloride.

Improving the heat tolerance of cotton is a major concern for breeding programs. To address this need, a fast and effect way of quantifying thermotolerant phenotypes is required. Triphenyl tetrazolium chloride (TTC) based enzyme viability testing following high-temperature stress can be used as a vegetative heat tolerance phenotype. This is because when live cells encounter a TTC solution, TTC undergoes a chemical reduction producing a visible, insoluble red product called triphenyl formazan, that can be quantified spectrophotometrically. However, existing TTC based cell viability assays cannot easily be deployed at the scale required in a crop improvement program. In this study, a heat stress assay (HSA) based on the use of TTC enzyme viability testing has been refined and improved for efficiency, reliability, and ease of use through four experiments. Sampling factors that may influence assay results, such as leaf age, plant water status, and short-term cold storage, were also investigated. Experiments conducted in this study have successfully downscaled the assay and identified an optimal sampling regime, enabling measurement of large segregating populations for application in breeding programs. The improved HSA methodology is important as it is proposed that long-term improvements in cotton thermotolerance can be achieved through the concurrent selection of superior phenotypes based on the HSA and yield performance in hot environments. Additionally, a new way of interpreting both heat tolerance and heat resistance was developed, differentiating genotypes that perform well at the time of a heat stress event and those that maintain a similar performance level to a non-stressed control.

Effects of elevated temperature and elevated CO2 on soil nitrification and ammonia-oxidizing microbial communities in field-grown crop.

Rising global air temperature and atmospheric CO2 are expected to have considerable effects on soil nutrient cycling and plant productivity. Soil nitrification controlled by ammonia-oxidizing bacteria and archaea (AOB and AOA) communities plays a key role in contributing to plant nitrogen (N) availability; however, response of soil nitrification and functional microbial communities to climate change and subsequent consequences for crop yields remain largely unknown. Cotton productivity is a function of temperature and N availability under well-watered conditions. In general, cotton growth responds positively to elevated CO2, but simultaneous warming may offset benefits of rising CO2. In this study, cotton was used as a model system to elucidate the short-term response of soil nitrification and ammonia-oxidizing communities to elevated temperature and elevated CO2 using field-based environmentally-controlled chambers. Elevated temperature (ambient + 1.1 °C) altered the AOA community, while elevated temperature and elevated CO2 (ambient + 132 ppm) significantly increased soil nitrification rate and shifted AOB and AOA communities, but these effects depended on cotton developmental stages. Ammonia-oxidizing community abundance and structure were statistically correlated with nitrifying activity. Our findings suggest that climate change will positively affect soil nitrifying communities, leading to an increase in process rates and subsequent N availability, which is directly linked to crop productivity.

Protecting cotton crops under elevated CO2 from waterlogging by managing ethylene.

Soil waterlogging and subsequent ethylene release from cotton (Gossypium hirsutum L.) tissues has been linked with abscission of developing cotton fruits. This glasshouse study investigates the effect of a 9-day waterlogging event and CO2 enrichment (eCO2, 700 parts per million (ppm)) on a fully linted cultivar 'Empire' and a lintless cotton mutant (5B). We hypothesised that cotton performance in extreme environments such as waterlogging can be improved through mitigating ethylene action. Plants were grown at 28:20°C day:night temperature, 50-70% relative humidity and a 14:10 light:dark photoperiod under natural light and were exposed to waterlogging and eCO2 at early reproductive growth. Ethylene synthesis was inhibited by spraying aminoethoxyvinylglycine (830ppm) 1 day before waterlogging. Waterlogging significantly increased ethylene release from both cotton genotypes, although fruit production was significantly inhibited only in Empire. Aminoethoxyvinylglycine consistently reduced waterlogging-induced abscission of fruits, mainly in Empire. Limited damage to fruits in 5B, despite increased ethylene production during waterlogging, suggested that fruit abscission in 5B was inhibited by disrupting ethylene metabolism genetically. Elevated CO2 promoted fruit production in both genotypes and was more effective in 5B than in Empire plants. Hence 5B produced more fruits than Empire, providing additional sinks (existing and new fruit) that enhanced the response to CO2 enrichment.

Cold plasma treatment for cotton seed germination improvement.

Adverse environmental conditions at planting, such as cold temperature or water limitation, can lead to a reduced level of seed germination and plant establishment for cotton. Cold atmospheric-pressure plasma (CAP) treatment of cotton seeds prior to planting may help alleviate this problem. CAP is ionised gas that has a range of biological activities due to the formation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show that a 27 minutes CAP treatment using air can significantly increase water absorption of the seed, and improve warm germination, metabolic chill test germination and chilling tolerance in cotton. We also observe that the beneficial effect of CAP treatment is long-lasting and stable as improved germination activity is still seen when treatment occurs 4 months before germination testing, suggesting that future large-scale industrial seed plasma treatments may still be effectively applied well (months) before the seed planting. We conclude that CAP treatment is a promising new tool for use in the cotton industry that has the potential to significantly improve plant establishment in a wider range of environmental conditions.

Endogenous Ethylene Concentration Is Not a Major Determinant of Fruit Abscission in Heat-Stressed Cotton (Gossypium hirsutum L.).

We investigated the role of ethylene in the response of cotton to high temperature using cotton genotypes with genetically interrupted ethylene metabolism. In the first experiment, Sicot 71BRF and 5B (a lintless variant with compromised ethylene metabolism) were exposed to 45°C, either by instantaneous heat shock or by ramping temperatures by 3°C daily for 1 week. One day prior to the start of heat treatment, half the plants were sprayed with 0.8 mM of the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG). In a subsequent experiment, Sicot 71BRF and a putatively heat-tolerant line, CIM 448, were exposed to 36 or 45°C for 1 week, and half the plants were sprayed with 20 µM of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, (ACC). High temperature exposure of plants in both experiments was performed at the peak reproductive phase (65-68 days after sowing). Elevated temperature (heat shock or ramping to 45°C) significantly reduced production and retention of fruits in all cotton lines used in this study. At the termination of heat treatment, cotton plants exposed to 45°C had at least 50% fewer fruits than plants under optimum temperature in all three genotypes, while plants at 36°C remained unaffected. Heat-stressed plants continued producing new squares (fruiting buds) after termination of heat stress but these squares did not turn into cotton bolls due to pollen infertility. In vitro inhibition of pollen germination by high temperatures supported this observation. Leaf photosynthesis (Pn) of heat-stressed plants (45°C) measured at the end of heat treatments remained significantly inhibited, despite an increased leaf stomatal conductance (gs), suggesting that high temperature impairs Pn independently of stomatal behavior. Metabolic injury was supported by high relative cellular injury and low photosystem II yield of the heat-stressed plants, indicating that high temperature impaired photosynthetic electron transport. Both heat shock and ramping of heat significantly reduced ethylene release from cotton leaf tissues measured at the end of heat treatment but modulating ethylene production via AVG or ACC application had no significant effect on fruit production or retention in heat-stressed cotton plants. Instead, high temperature accelerated fruit abortion by impairing pollen development and/or restricting leaf photosynthesis.

Warming alters the positive impact of elevated CO2 concentration on cotton growth and physiology during soil water deficit.

Alterations in climate factors such as rising CO2 concentration ([CO2]), warming and reduced precipitation may have significant impacts on plant physiology and growth. This research investigated the interactive effects of elevated [CO2], warming and soil water deficit on biomass production, leaf-level physiological responses and whole-plant water use efficiency (WUEP) in cotton (Gossypium hirsutum L.). Cotton was grown in the glasshouse under two [CO2] treatments (CA, 400µLL-1; CE, 640µLL-1) and two temperature treatments (TA, 28°C:17°C day:night; TE, 32°C:21°C day:night). Plants were subjected to two progressive water deficit cycles, with a 5-day recovery period between the water deficit periods. CE increased vegetative biomass and photosynthetic rates, and decreased stomatal conductance in TA; however, these responses to CE were not evident under TE. CE increased whole-plant water loss under TA, but increased WUEp, whereas increased whole-plant water loss in TE decreased WUEp regardless of atmospheric [CO2]. CE may provide some positive growth and physiological benefits to cotton at TA if sufficient water is available but CE will not mitigate the negative effects of rising temperature on cotton growth and physiology in future environments.

The effect of elevated atmospheric [CO2] and increased temperatures on an older and modern cotton cultivar.

Changes in atmospheric [CO2], temperature and precipitation under projected climate change scenarios may have significant impacts on the physiology and yield of cotton. Understanding the implications of integrated environmental impacts on cotton is critical for developing cotton systems that are resilient to stresses induced by climate change. The objective of this study was to quantify the physiological and growth capacity of two cotton cultivars under current and future climate regimes. This experiment compared the early-season growth and physiological response of an older (DP16, released in the 1970s) and a modern (Sicot 71BRF, released in 2008) cotton cultivar grown in ambient and elevated atmospheric [CO2] (CA, 400µLL-1 and CE, 640µLL-1 respectively) and two temperature (TA, 28/17°C and TE, 32/21°C, day/night, respectively) treatments under well-watered conditions. CE increased biomass and photosynthetic rates compared with CA, and TE increased plant biomass. Although limited by the comparison of one older and one modern cultivar, our results suggest that substantial potential may exist to increase breeding selection of cotton cultivars that are responsive to both TE and CE.

Consequences of waterlogging in cotton and opportunities for mitigation of yield losses.

Climatic variability, typified by erratic heavy-rainfall events, causes waterlogging in intensively irrigated crops and is exacerbated under warm temperature regimes on soils with poor internal drainage. Irrigated cotton is often grown in precisely these conditions, exposing it to waterlogging-induced yield losses after substantial summer rainfall. This calls for a deeper understanding of mechanisms of waterlogging tolerance and its relevance to cotton. Hence this review suggests possible causes of waterlogging-induced yield loss in cotton and approaches to improvement of waterlogging tolerance, drawing upon the slight body of published data in cotton and principles from other species. The yield penalty depends on soil type, phenological stage and cumulative period of root exposure to air-filled porosities below 10 %. Events in the soil include O2 deficiency in the root zone that changes the redox state of nutrients, making them unavailable (e.g. nitrogen) or potentially toxic for plants. Furthermore, root-derived hormones that are transported in the xylem have long been associated with oxygen deficits. These belowground effects (impaired root growth, nutrient uptake and transport, hormonal signalling) affect the shoots, interfering with canopy development, photosynthesis and radiation-use efficiency. Compared with the more waterlogging-tolerant cereals, cotton does not have identified adaptations to waterlogging in the root zone, forming no conspicuous root aerenchyma and having low fermentative activity. We speculate that these factors contribute substantially to the sensitivity of cotton to sustained periods of waterlogging. We discuss the impact of these belowground factors on shoot performance, photosynthesis and yield components. Management practices, i.e. soil aeration, scheduling irrigation and fertilizer application, can reduce waterlogging-induced damage. Limiting ethylene biosynthesis using anti-ethylene agents and down-regulating expression of genes controlling ethylene biosynthesis are strong candidates to minimize yield losses in waterlogged cotton crops. Other key pathways of anoxia tolerance are also cited as potential tools towards waterlogging-tolerant cotton genotypes.

Understanding the molecular events underpinning cultivar differences in the physiological performance and heat tolerance of cotton (Gossypium hirsutum).

Diurnal or prolonged exposure to air temperatures above the thermal optimum for a plant can impair physiological performance and reduce crop yields. This study investigated the molecular response to heat stress of two high-yielding cotton (Gossypium hirsutum L.) cultivars with contrasting heat tolerance. Using global gene profiling, 575 of 21854 genes assayed were affected by heat stress, ~60% of which were induced. Genes encoding heat shock proteins, transcription factors and protein cleavage enzymes were induced, whereas genes encoding proteins associated with electron flow, photosynthesis, glycolysis, cell wall synthesis and secondary metabolism were generally repressed under heat stress. Cultivar differences for the expression profiles of a subset of heat-responsive genes analysed using quantitative PCR over a 7-h heat stress period were associated with expression level changes rather than the presence or absence of transcripts. Expression differences reflected previously determined differences for yield, photosynthesis, electron transport rate, quenching, membrane integrity and enzyme viability under growth cabinet and field-generated heat stress, and may explain cultivar differences in leaf-level heat tolerance. This study provides a platform for understanding the molecular changes associated with the physiological performance and heat tolerance of cotton cultivars that may aid breeding for improved performance in warm and hot field environments.

Developing controlled environment screening for high-temperature tolerance in cotton that accurately reflects performance in the field.

In this study we investigated the heat tolerance of high yielding Australian cotton (Gossypium hirsutum L.) cultivars using a multi-level approach encompassing physiological assays and measurements of performance. Two cultivars with known field performance were evaluated for heat tolerance under optimal (32°C) and high (42°C) temperatures in a growth cabinet with a cell membrane integrity assay. Impacts of temperature on growth were evaluated with leaf level measurements of gas exchange and chlorophyll fluorescence. To extend the multi-level approach, the expression of a Rubisco activase regulating gene (GhRCAα2) was also determined. Consistent with previously determined differences in the field, cultivar Sicot 53 outperformed Sicala 45 for the cell membrane integrity assay; this finding was reflective of cultivar differences in gas exchange and chlorophyll fluorescence. Cultivar differences were also consistent for expression of GhRCAα2, which may also help explain differences in physiological performance, particularly photosynthesis. This study reaffirmed that physiological and molecular assays were sufficiently sensitive to resolve genotypic differences in heat tolerance and that these differences translate to physiological performance. By comparing performance under high temperatures in the growth cabinet and field, this approach validates the use of rapid screening tools in conjunction with a multi-level approach for heat tolerance detection.