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Advancements in hyperspectral imaging (HSI) and establishment of dedicated plant phenotyping facilities have enabled researchers to gather large quantities of plant spectral images with the aim of inferring target phenotypes non-destructively. However, large volumes of data that result from HSI and corequisite specialized methods for analysis may prevent plant scientists from taking full advantage of these systems. Here, we explore estimation of physiological traits in 23 rice accessions using an automated HSI system. Under contrasting nitrogen conditions, HSI data are used to classify treatment groups with ≥ 83% accuracy by utilizing support vector machines. Out of the 14 physiological traits collected, leaf-level nitrogen content (N, %) and carbon to nitrogen ratio(C:N)could also be predicted from the hyperspectral imaging data with normalized root mean square error of predictions smaller than 14% (R2of 0.88 forNand 0.75 forC:N).This study demonstrates the potential of using an automated HSI system to analyze genotypic variation for physiological traits in a diverse panel of rice; to help lower barriers of application of hyperspectral imaging in the greater plant science research community, analysis scripts used in this study are carefully documented and made publicly available.HIGHLIGHTData from an automated hyperspectral imaging system are used to classify nitrogen treatment and predict leaf-level nitrogen content and carbon to nitrogen ratio during vegetative growth in rice.

Crop improvement is crucial to ensuring global food security under climate change, and hence there is a pressing need for phenotypic observations that are both high throughput and improve mechanistic understanding of plant responses to environmental cues and limitations. In this study, chlorophyll a fluorescence light response curves and gas-exchange observations are combined to test the photosynthetic response to moderate drought in four genotypes of Brassica rapa. The quantum yield of PSII (ϕ(PSII)) is here analyzed as an exponential decline under changing light intensity and soil moisture. Both the maximum ϕ(PSII) and the rate of ϕ(PSII) decline across a large range of light intensities (0–1,000 μmol photons m(−2) s(−1); β(PSII)) are negatively affected by drought. We introduce an alternative photosynthesis model (β(PSII) model) incorporating parameters from rapid fluorescence response curves. Specifically, the model uses β(PSII) as an input for estimating the photosynthetic electron transport rate, which agrees well with two existing photosynthesis models (Farquhar-von Caemmerer-Berry and Yin). The β(PSII) model represents a major improvement in photosynthesis modeling through the integration of high-throughput fluorescence phenotyping data, resulting in gained parameters of high mechanistic value.

Phaseolus vulgaris L. plants were irradiated with different doses (0.3, 10, 50 and 100 Gy) of X-rays in order to obtain a reference curve of response to ionizing radiations for this species. Growth analysis, gas exchange and chlorophyll a fluorescence measurements were performed to estimate the radio-resistance of bean plants. Specifically, there was a negative influence of X-rays on the net photosynthesis rate at 50 and 100 Gy, already on the day of irradiation. Experimental data showed a recovery over time in the gas exchange while the theoretical maximum photochemical efficiency of the photosystem II (Fv/Fm) was fairly constant throughout the period of measurements (20 days) and for all the experimental conditions. On the other hand, the quantum yield of PSII linear electron transport (ΦPSII) and non-photochemical quenching (NPQ) were deeply influenced over time by X-ray dose, suggesting a decrease in the functionality of the photosynthetic apparatus at the highest radiation doses. The growth was affected only at the highest doses of radiation with a significant and severe reduction of leaf expansion and number of leaves per plant. Despite the arrest in growth, X-ray exposure seems to trigger an increased photochemical activity probably signifying that P. vulgaris plants have a fairly elevated resistance to this kind of ionizing radiation. Our current results will provide a complete analysis of the photosystem II (PSII) response of P. vulgaris to different doses (0.3, 10, 50 and 100 Gy) of X-rays, providing sound references for both space-oriented and radioecology questions.

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii , the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii . The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis -regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis -regulatory CrCO-binding sites at key photoprotection genes.

Vegetation controls carbon and water fluxes because of the fundamental tradeoff between carbon dioxide uptake and water loss occurring when stomata are open. Quantifying the rates of this exchange typically requires either intensive gas exchange or destructive harvesting of tissues and mass spectrometry analyses. Recent developments in high-throughput methods have enhanced our capacity to empirically test plant-environmental interactions. The vast integration characterizing satellite remote sensing methods masks organ-level physiological mechanisms limiting the predictive capability of current process models. Hence, more ground truth studies are necessary to determine the amount of mechanistic information needed to improve our understanding of forest, crop, and land management. Imaging methodologies, such as thermal and chlorophyll a fluorescence, are currently used to collect information for relevant traits such as water use, growth, and stress response. We tested these techniques during progressive drought across species with different susceptibility in controlled greenhouse conditions. We chose two highly represented tree species in North America: the gymnosperm Pinus ponderosa and the angiosperm Populus tremuloides. To better explore the whole drought response parameter space, we also tested a crop (Brassica rapa) and desert shrub (Artemisia tridentata). Thermal and fluorescence images of the canopy were coupled with leaf-level measurements as we performed three tests to predict drought response using 1) leaf temperature, 2) chlorophyll a fluorescence, and 3) the combination of the two. At five days of drought, leaf temperature increased 7 and 10%, accounting for 63% and 73% of the variation in stomatal conductance for both tree species, respectively. The fluorescence signal from images decreased ~12% and ~83% in moderately and severely droughted leaves respectively, reaching zero at mortality. Leaf water status was then predicted using a Bayesian approach that incorporated measurements’ uncertainty and parsimony in the analysis of the parameters. Changes in canopy temperature provided confident predictions for the reductions of daily evapotranspiration at the onset of drought. Empirically combining thermal and fluorescence measurements improved predictions (R2 = 0.81) of midday leaf water potential compared to univariate models. Our results represent an important step towards quantifying plant water status during drought using first principles that do not require species-specific information.

The circadian clock is a molecular timekeeper that matches endogenous rhythms in diverse traits with 24-h cycles in the external environment. Although a lack of clock resonance to the environment is detrimental to performance, clock phenotypes in wild populations nevertheless deviate substantially from the predicted optimal cycle length of 24 h, and significant genetic variation exists for circadian parameters. Here, we describe covariation between 2 traits considered to reflect adaptation to different aspects of temporal environmental heterogeneity, circadian rhythms (adaptation to daily environmental cycles) and flowering time (adaptation to seasonal cycles), in a Rocky Mountain population of the mustard Boechera stricta, a North American relative of Arabidopsis thaliana. We found that 18 families that differ in circadian period in leaf movement by 3.5 h expressed genetic diversity in first-year growth, reproductive phenology, vegetative size at reproduction, and starch concentration following vernalization. The families exhibited a large (~90-day) range in mean flowering time, even though the spatial scale of population sampling covered only a few hundred meters. Circadian period covaried with other traits such that longer-period families flowered earlier and at a larger size, a trait combination predicted to yield a fitness benefit in the wild. Circadian clock research in model systems has previously shown that mutations in clock genes influence phenology. Our results widen the scope of this research by illustrating a link between naturally segregating clock variation and reproductive phenology among wild genotypes, suggesting that the causes of genetic diversity in the clock lie partly in adaptation to seasonal environmental heterogeneity.

Climate models predict widespread increases in both drought intensity and duration in the next decades. Although water deficiency is a significant determinant of plant survival, limited understanding of plant responses to extreme drought impedes forecasts of both forest and crop productivity under increasing aridity. Drought induces a suite of physiological responses; however, we lack an accurate mechanistic description of plant response to lethal drought that would improve predictive understanding of mortality under altered climate conditions. Here, proxies for leaf cellular damage, chlorophyll a fluorescence, and electrolyte leakage were directly associated with failure to recover from drought upon rewatering in Brassicarapa (genotype R500) and thus define the exact timing of drought-induced death. We validated our results using a second genotype (imb211) that differs substantially in life history traits. Our study demonstrates that whereas changes in carbon dynamics and water transport are critical indicators of drought stress, they can be unrelated to visible metrics of mortality, i.e. lack of meristematic activity and regrowth. In contrast, membrane failure at the cellular scale is the most proximate cause of death. This hypothesis was corroborated in two gymnosperms (Picea engelmannii and Pinus contorta) that experienced lethal water stress in the field and in laboratory conditions. We suggest that measurement of chlorophyll a fluorescence can be used to operationally define plant death arising from drought, and improved plant characterization can enhance surface model predictions of drought mortality and its consequences to ecosystem services at a global scale.

Significance Photoprotection is an indispensable component of abiotic stress tolerance in all photosynthetic organisms. In contrast to land plants, the transcript abundance of photoprotection genes is strongly controlled by light in the model green alga Chlamydomonas reinhardtii. Here we show that the repression of photoprotection genes in low light depends on 2 putative components of a conserved E3 ubiquitin ligase complex. We also show that this regulation of gene expression depends on a transcription factor that is conserved among plants and algae and that acts downstream of the putative E3 ubiquitin ligase complex. Our work provides mechanistic insight into how a deeply conserved light signaling pathway is wired differently in C. reinhardtii, giving rise to light control of photoprotection gene expression., Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii. The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes.

The total solar eclipse of August 21, 2017 created a path of totality ~115 km in width across the United States. While eclipse observations have shown distinct responses in animal behavior often emulating nocturnal behavior, the influence of eclipses on plant physiology are less understood. We investigated physiological perturbations due to rapid changes of sunlight and air temperature in big sagebrush (Artemisia tridentata ssp. vaseyana), a desert shrub common within the path of eclipse totality. Leaf gas exchange, water potential, and chlorophyll a fluorescence were monitored during the eclipse and compared to responses obtained the day before in absence of the eclipse. On the day of the eclipse, air temperature decreased by 6.4 °C, coupled with a 1.0 kPa drop in vapor pressure deficit having a 9-minute lag following totality. Using chlorophyll a fluorescence measurements, we found photosynthetic efficiency of photosystem II (Fv’/Fm’) recovered to near dark acclimated state (i.e., 87%), but the short duration of darkness did not allow for complete recovery. Gas exchange data and a simple light response model were used to estimate a 14% reduction in carbon assimilation for one day over sagebrush dominated areas within the path of totality for the Western United States.

Process-based models of plant productivity provide a means of yield prediction that can inform best practices in agriculture and land management. However, current biophysical models fail in capturing both genotypic and phenotypic variation under changing environmental conditions. Physiological traits affecting final yield and ecosystem productivity are the result of gene expression, protein translation, and metabolite formation which are controlled by specific alleles acting alone and in response to time and environmental cues. Biophysical interactions take place simultaneously across several scales of time and space, giving rise to high levels of complexity and limiting the predictive capacity of existing analytical approaches. While statistical methods can partially quantify genotype-by-environment connections, biophysical process models miss exploring genotypic diversity and they cannot currently be implemented with -omics data, i.e. the entire collection of entities such as gene transcripts, metabolites, and proteins. Here we present a novel framework that utilizes Bayesian networks to provide probabilities that link gene expression levels to trait occurrence. We propose the use of these gained probabilities to inform parameters of existing biophysical models. Merging transcriptomic information into process-based models allows for greater integration of empirical data and processes across different scales in complex systems, while increasing the likelihood of growth predictions under unknown environmental conditions.

A growing body of evidence demonstrates a significant relationship between cellular redox state and circadian rhythms. Each day these two vital components of plant biology influence one another, dictating the pace for metabolism and physiology. Diverse environmental stressors can disrupt this condition and, although plant scientists have made significant progress in re-constructing functional networks of plant stress responses, stress impacts on the clock-redox crosstalk is poorly understood. Inter-connected phenomena such as redox state and metabolism, internal and external environments, cellular homeostasis and rhythms can impede predictive understanding of coordinated regulation of plant stress response. The integration of circadian clock effects into predictive network models is likely to increase final yield and better predict plant responses to stress. To achieve such integrated understanding, it is necessary to consider the internal clock not only as a gatekeeper of environmental responses but also as a target of stress syndromes. Using chlorophyll fluorescence as a reliable and high-throughput probe of stress coupled to functional genomics and metabolomics will provide insights on the crosstalk across a wide range of stress severity and duration, including potential insights into oxidative stress response and signaling. We suggest the efficiency of photosystem II in light conditions (Fv′/Fm′) to be the most dynamic of the fluorescence variables and therefore the most reliable parameter to follow the stress response from early sensing to mortality.

The circadian clock regulates many aspects of leaf gas supply and biochemical demand for CO2 20 , and is hypothesized to improve plant performance. Yet the extent to which the clock may regulate the efficiency of photosystem II (PSII) and photoprotective mechanisms such as heat dissipation remains largely unexplored. Based on measurements of chlorophyll a fluorescence, we estimated the maximum efficiency of photosystem II in light (Fv'/Fm') and heat dissipation by non-photochemical quenching (NPQ). We further dissected total NPQ into its main components, qE (pH-dependent quenching), qT (state-transition quenching) and qI (quenching related to photoinhibition), in clock mutant genotypes of Arabidopsis thaliana, the cognate wild-type genotypes, and a panel of recombinant inbred lines (RILs) expressing quantitative variation in clock period. Compared to mutants with altered clock function, we observed that wild-type genotypes with clock period lengths of approximately 24 hr had both higher levels of Fv'/Fm ', indicative of improved PSII function, and reduced NPQ, suggestive of lower stress on PSII light harvesting complexes. In the RILs, genetic variances were significant for Fv'/Fm' and all three components of NPQ, with qE explaining the greatest proportion of NPQ. Bivariate tests of association and structural equation models of hierarchical trait relationships showed that quantitative clock variation was empirically associated with Fv'/Fm' and NPQ, with qE mediating the relationship with gas exchange. The results demonstrate significant segregating variation for all photoprotective components, and suggest the adaptive significance of the clock may partly derive from its regulation of the light reactions of photosynthesis and of photoprotective mechanisms. Key words: Arabidopsis thaliana, circadian rhythms, chlorophyll a fluorescence, maximum efficiency of PSII, non-photochemical quenching

Based on the examination and quantitative comparison of the approaches used to assess the energy partitioning in photosystem II, the unified method was proposed to calculate the contribution of the components of nonphotochemical quenching.

In highlight stress conditions, the mechanism of non-photochemical quenching (NPQ) of chlorophyll fluorescence is triggered at the chloroplast level. This process allows thermal quenching of the excessive excitation energy and it is strictly related to the efficiency of the xanthophyll cycle. Nowadays, the utilization of the nuclear magnetic resonance (NMR) spectroscopy provides a powerful complementary way for the identification and quantitative analysis of plant metabolites either in vivo or in tissue extracts. Seeing that the oxidative damage caused by light stress in plants and the consequent involvement of pigments are widely studied, NMR spectroscopy can be utilized to compare crude leaf extract at different levels of light stress, allowing an analysis of these compounds. In this paper, the identification of possible relationships between light stress and 1H NMR signal variations is discussed. The analysis of the 1H NMR (1D) spectra of two agronomic species (Spinacia oleracea and Beta vulgaris) exposed to different light intensities is presented. In particular, change in carotenoids and xanthophylls signals are analyzed.

Non-photochemical quenching (NPQ) of chlorophyll a fluorescence and leaf gas-exchange were investigated in relation to the chlorophyll and carotenoid content, and the xanthophyll cycles in wild type tomato (Solanum lycopersicum, L. cv Red Setter (RS)) and in two transgenic lines (UO and UU) over-expressing β-carotene hydroxylase. Potted plants were grown in a glasshouse under low light (LL, 100 μmol m−2 s−1) or high light (HL, 300 μmol m−2 s−1). The maximum quantum efficiency of photosystems II (PSII) photochemistry in dark-adapted leaves (Fv/Fm) was higher than 0.82 in all treatments while photosynthetic CO2 assimilation (A) was higher than 14 μmol m−2 s−1, and stomatal conductance (gs) higher than 0.4 mol m−2 s−1 in HL plants, indicating no effects induced by the genetic modification. Chlorophyll content and composition changed little, whereas transgenic plants had up to 47% higher total carotenoid content than wild type plants. Violaxanthin was the most abundant carotenoid in transgenic plants, with more than 2-fold higher content than the average 0.586 mg g−1 found in RS plants. Transgenic plants had similar light-induced steady-state NPQ compared to wild type plants, but had slower dark relaxation because of the decreased de-epoxydation state index due to the higher violaxanthin accumulation, despite the higher zeaxanthin content.

Non-photochemical quenching (NPQ) is a complex and still unclear mechanism essential for higher plants. The intensive research on this subject has highlighted three main components of NPQ: energy-dependent process (qE); state transitions to balance the excitation of PSII and PSI (qT); and photoinhibitory processes (qI). Recently, these components have been resolved as quantum yields according to the energy partitioning approach that takes into account the rate constants of every process involved in the quenching mechanisms of excited chlorophylls. In this study a fully extended quantum yield approach and the introduction of novel equations to assess the yields of each NPQ component are presented. Furthermore, a complete analysis of the yield of NPQ in Beta vulgaris exposed to different irradiances has been carried out. In agreement with experimental results here it is shown that the previous approach may amplify the yield of qE component and flatten the quantitative results of fluorescence analysis. Moreover, the significance of taking into account the physiological variability of NPQ for a correct assessment of energy partitioning is demonstrated.

Non-photochemical quenching (NPQ) was measured in Col-0 and npq1 mutant plants of Arabidopsis thaliana exposed for 40 min to different light intensities (85, 175, 420, 750 and 1,200 μmol photons m−2 s−1). A light-induced increase of NPQ was measured in Col-0 as well as in npq1 plants but no significant differences were found between two genotypes. The three mechanisms of quenching (qE, qT and qI) were determined by the analysis of the dark relaxation kinetics of NPQ. In most environmental conditions of this work, qE was not the major component of NPQ except in Col-0 plants at 1,200 µmol photons m−2 s−1. At low light intensities qT was the sole component of NPQ and it increased at higher irradiances as absolute value both genotypes.