On the concepts and correct use of radiometric quantities for assessing the light environment and their application to plant research.

Light is one of the most important factors for photosynthetic organisms to grow. Historically, the amount of light in plant sciences has been referred to as light intensity, irradiance, photosynthetic active radiation, photon flux, photon flux density, etc. On occasion, all these terms are used interchangeably, yet they refer to different physical units and each metric offers distinct information. Even for experts in the fields of plant photobiology, the use of these terms is confusing, and there is a loose implementation of each concept. This makes the use of radiometric units even more confusing to non-experts when looking for ways to measure light, since they could easily feel overwhelmed by the specialized literature. The use of scientific concepts must be accurate, as ambiguity in the use of radiometric quantities can lead to inconsistencies in analysis, thus decreasing the comparability between experiments and to the formulation of incorrect experimental designs. In this review, we provide a simple yet comprehensive view of the use of radiometric quantities in an effort to clarify their meaning and applications. To facilitate understanding, we adopt a minimum amount of mathematical expressions and provide a historical summary of the use of radiometry (with emphasis on plant sciences), examples of uses, and a review of the available instrumentation for radiometric measurements.

Phenomics: conceptualization and importance for plant physiology.

Phenomics is a relatively new discipline of biology that has been widely applied in several fields, mainly in crop sciences. We reviewed the concepts used in this discipline (particularly for plants) and found a lack of consensus on what defines a phenomic study. Furthermore, phenomics has been primarily developed around its technical aspects (operationalization), while the conceptual framework of the actual research lags behind. Each research group has given its own interpretation of this 'omic' and thus unwittingly created a 'conceptual controversy'. Addressing this issue is of particular importance, as the experimental designs and concepts of phenomics are so diverse that it is difficult to compare studies. In this opinion article, we evaluate the conceptual framework of phenomics.

Good vibrations: Raman spectroscopy enables insights into plant biochemical composition.

Non-invasive techniques are needed to enable an integrated understanding of plant metabolic responses to environmental stresses. Raman spectroscopy is one such technique, allowing non-destructive chemical characterisation of samples in situ and in vivo and resolving the chemical composition of plant material at scales from microns to metres. Here, we review Raman band assignments of pigments, structural and non-structural carbohydrates, lipids, proteins and secondary metabolites in plant material and consider opportunities this technology raises for studies in vascular plant physiology.

Advanced technologies in studying plant photosynthesis: principles and applications.

sion="1.0" encoding="utf-8"?> FP Functional Plant Biology Funct. Plant Biol. 1445-4408 1445-4416 CSIRO Publishing 36 Gardiner Road Clayton VIC 3168 Australia FP22050 10.1071/FP22050 Foreword Advanced technologies in studying plant photosynthesis: principles and applications A. Zavafer et al . https://orcid.org/0000-0002-8905-1618 Zavafer Alonso A Fan Dayong B * https://orcid.org/0000-0001-8150-9535 Murakami Keach C Handling Editor Shabala Sergey Plant Science Division, Research School of Biology, The Australian National University, Canberra, ACT 2001, Australia. Hokkaido Agricultural Research Center (HARC), National Agriculture and Food Research Organization (NARO), 1 Hitsujigaoka, Toyohira, Sapporo 062-8555, Japan. College of Forestry, Beijing Forestry, University, Beijing 100083, China. * Correspondence to: Dayong Fan Hokkaido Agricultural Research Center (HARC), National Agriculture and Food Research Organization (NARO), 1 Hitsujigaoka, Toyohira, Sapporo 062-8555, Japan Email: dayong73fan@163.com 9 May 2022 49 6 Special Issue i iii 9 May 2022 Published: 9 May 2022 © 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing 2022 The Authors The foreword to this special issue on the advanced technologies in studying photosynthesis focuses on the main contributions of Fred Chow, one of the key Australian scientists studying light reactions in plants.

Corrigendum to: Investigating the impact of light quality on macromolecular of Chaetoceros muelleri.

Diatoms (Bacillariophyceae) are important to primary productivity of aquatic ecosystems. This algal group is also a valuable source of high value compounds that are utilised as aquaculture feed. The productivity of diatoms is strongly driven by light and CO2 availability, and macro- and micronutrient concentrations. The light dependency of biomass productivity and metabolite composition is well researched in diatoms, but information on the impact of light quality, particularly the productivity return on energy invested when using different monochromatic light sources, remains scarce. In this work, the productivity return on energy invested of improving growth rate, photosynthetic activity, and metabolite productivity of the diatom Chaetoceros muelleri under defined wavelengths (blue, red, and green) as well as while light is analysed. By adjusting the different light qualities to equal photosynthetically utilisable radiation, it was found that the growth rate and photosynthetic oxygen evolution was unchanged under white, blue, and green light, but it was lower under red light. Blue light improved the productivity return on energy invested for biomass, total protein, total lipid, total carbohydrate, and in fatty acids production, which would suggest that blue light should be used for aquaculture feed production.

Fred Chow: the contributions of a quiet giant of photoinhibition and photoprotection.

Wah Soon (Fred) Chow has been a major contributor to photosynthesis research since the late 20th century. Fred, a quiet, gentle, smart and prolific writer, has contributed to our understanding of thylakoid structure, cyclic electron flow and the development of novel methods for phenotyping plants. However, a third of his productivity centres on the understanding of photoinhibition and photoprotection, which we honour herein. We give a brief biographical account of his academic trajectory, followed by a chronological and conceptual summary of his contributions to the field of photodamage and photoprotection. We thereby hope to introduce the work of Fred to young readers and non-experts in the field of photoinhibition.

Investigating the impact of light quality on macromolecular composition of Chaetoceros muelleri.

Diatoms (Bacillariophyceae) are important to primary productivity of aquatic ecosystems. This algal group is also a valuable source of high value compounds that are utilised as aquaculture feed. The productivity of diatoms is strongly driven by light and CO2 availability, and macro- and micronutrient concentrations. The light dependency of biomass productivity and metabolite composition is well researched in diatoms, but information on the impact of light quality, particularly the productivity return on energy invested when using different monochromatic light sources, remains scarce. In this work, the productivity return on energy invested of improving growth rate, photosynthetic activity, and metabolite productivity of the diatom Chaetoceros muelleri under defined wavelengths (blue, red, and green) as well as while light is analysed. By adjusting the different light qualities to equal photosynthetically utilisable radiation, it was found that the growth rate and photosynthetic oxygen evolution was unchanged under white, blue, and green light, but it was lower under red light. Blue light improved the productivity return on energy invested for biomass, total protein, total lipid, total carbohydrate, and in fatty acids production, which would suggest that blue light should be used for aquaculture feed production.

A theoretical framework of the hybrid mechanism of photosystem II photodamage.

Photodamage of photosystem II is a significant physiological process that is prevalent in the fields of photobiology, photosynthesis research and plant/algal stress. Since its discovery, numerous efforts have been devoted to determine the causes and mechanisms of action of photosystem II photodamage. There are two contrasting hypotheses to explain photodamage: (1) the excitation pressure induced by light absorption by the photosynthetic pigments and (2) direct photodamage of the Mn cluster located at the water-splitting site, which is independent of excitation pressure. While these two hypotheses seemed mutually exclusive, during the last decade, several independent works have proposed an alternative approach indicating that both hypotheses are valid. This was termed the dual hypothesis of photosystem II photodamage, and it postulates that both excess excitation and direct Mn photodamage operate at the same time, independently or in a synergic manner, depending on the type of sample, temperature, light spectrum, or other environmental stressors. In this mini-review, a brief summary of the contrasting hypotheses is presented, followed by recapitulation of key discoveries in the field of photosystem II photodamage of the last decade, and a synthesis of how these works support a full hybrid framework (operation of several mechanisms and their permutations) to explain PSII photodamage. All these are in recognition of Prof. Wah Soon Chow (the Australian National University), one of the key proposers of the dual hypothesis.

Wah Soon Chow, a teacher, a friend and a colleague.

To finish this special issue, some friends, colleagues and students of Prof. Chow (Emeritus Professor, the Research School of Biology, the Australian National University) have written small tributes to acknowledge not only his eminent career but to describe his wonderful personality.

Effects of MPK3 and MPK6 kinases on the chloroplast architecture and function induced by cold acclimation in Arabidopsis.

Exposure to low, non-freezing temperatures develops freezing tolerance in many plant species. Such process is called cold acclimation. Molecular changes undergone during cold acclimation are orchestrated by signalling networks including MAP kinases. Structure and function of chloroplasts are affected by low temperatures. The aim of this work was to study how the MAP kinases MPK3 and MPK6 are involved in the chloroplast performance upon a long period of cold acclimation. We used Arabidopsis thaliana wild type and mpk3 and mpk6 mutants. Adult plants were acclimated during 7 days at 4 °C and then measurements of PSII performance and chloroplast ultrastructure were carried out. Only the mpk6 acclimated plants showed a high freezing sensitivity. No differences in the PSII function were observed in the plants from the three genotypes exposed to non-acclimated or acclimated conditions. The acclimation of wild-type plants produced severe alterations in the ultrastructure of chloroplast and thylakoids, which was more accentuated in the mpk plants. However, only the mpk6 mutant was unable to internalize the damaged chloroplasts into the vacuole. These results indicate that cold acclimation induces alterations in the chloroplast architecture leading to preserve an optimal performance of PSII. MPK3 and MPK6 are necessary to regulate these morphological changes, but besides, MPK6 is needed to the vacuolization of the damaged chloroplasts, suggesting a role in the chloroplast recycling during cold acclimation. The latter could be quite relevant, since it could explain why this mutant is the only one showing an extremely low freezing tolerance.

Foliar water uptake via cork warts in mangroves of the Sonneratia genus.

Foliar water uptake (FWU) occurs in plants of diverse ecosystems; however, the diversity of pathways and their associated FWU kinetics remain poorly resolved. We characterized a novel FWU pathway in two mangrove species of the Sonneratia genus, S. alba and S. caseolaris. Further, we assessed the influence of leaf wetting duration, wet-dry seasonality and leaf dehydration on leaf conductance to surface water (Ksurf ). The symplastic tracer dye, disodium fluorescein, revealed living cells subtending and encircling leaf epidermal structures known as cork warts as a pathway of FWU entry into the leaf. Rehydration kinetics experiments revealed a novel mode of FWU, with slow and steady rates of water uptake persistent over a duration of 12 hr. Ksurf increased with longer durations of leaf wetting and was greater in leaves with more negative water potentials at the initiation of leaf wetting. Ksurf declined by 68% between wet and dry seasons. Our results suggest that FWU via cork warts in Sonneratia sp. may be rate limited and under active regulation. We conclude that FWU pathways in halophytes may require ion exclusion to avoid uptake of salt when inundated, paralleling the capacity of halophyte roots for ion selectivity during water acquisition.

Organized Disassembly of Photosynthesis During Programmed Cell Death Mediated By Long Chain Bases.

In plants, pathogen triggered programmed cell death (PCD) is frequently mediated by polar lipid molecules referred as long chain bases (LCBs) or ceramides. PCD interceded by LCBs is a well-organized process where several cell organelles play important roles. In fact, light-dependent reactions in the chloroplast have been proposed as major players during PCD, however, the functional aspects of the chloroplast during PCD are largely unknown. For this reason, we investigated events that lead to disassembly of the chloroplast during PCD mediated by LCBs. To do so, LCB elevation was induced with Pseudomonas syringae pv. tomato (a non-host pathogen) or Fumonisin B1 in Phaseolus vulgaris. Then, we performed biochemical tests to detect PCD triggering events (phytosphingosine rises, MPK activation and H2O2 generation) followed by chloroplast structural and functional tests. Observations of the chloroplast, via optical phenotyping methods combined with microscopy, indicated that the loss of photosynthetic linear electron transport coincides with the organized ultrastructure disassembly. In addition, structural changes occurred in parallel with accumulation of H2O2 inside the chloroplast. These features revealed the collapse of chloroplast integrity and function as a mechanism leading to the irreversible execution of the PCD promoted by LCBs.

Global Trends of Usage of Chlorophyll Fluorescence and Projections for the Next Decade.

Chlorophyll fluorescence is the most widely used set of techniques to probe photosynthesis and plant stress. Its great versatility has given rise to different routine methods to study plants and algae. The three main technical platforms are pulse amplitude modulation (PAM), fast rise of chlorophyll fluorescence, and fast repetition rate. Solar-induced fluorescence (SIF) has also gained interest in the last few years. Works have compared their advantages and their underlying theory, with many arguments advanced as to which method is the most accurate and useful. To date, no data has assessed the exact magnitude of popularity and influence for each methodology. In this work, we have taken the bibliometrics of the past decade for each of the four platforms, have evaluated the public scientific opinion toward each method, and possibly identified a geographical bias. We used various metrics to assess influence and popularity for the four routine platforms compared in this study and found that, overall, PAM currently has the highest values, although the more recent SIF has increased in popularity rapidly during the last decade. This indicates that PAM is currently one of the fundamental tools in chlorophyll fluorescence.

Two Quenchers Formed During Photodamage of Phostosystem II and The Role of One Quencher in Preemptive Photoprotection.

The quenching of chlorophyll fluorescence caused by photodamage of Photosystem II (qI) is a well recognized phenomenon, where the nature and physiological role of which are still debatable. Paradoxically, photodamage to the reaction centre of Photosystem II is supposed to be alleviated by excitation quenching mechanisms which manifest as fluorescence quenchers. Here we investigated the time course of PSII photodamage in vivo and in vitro and that of picosecond time-resolved chlorophyll fluorescence (quencher formation). Two long-lived fluorescence quenching processes during photodamage were observed and were formed at different speeds. The slow-developing quenching process exhibited a time course similar to that of the accumulation of photodamaged PSII, while the fast-developing process took place faster than the light-induced PSII damage. We attribute the slow process to the accumulation of photodamaged PSII and the fast process to an independent quenching mechanism that precedes PSII photodamage and that alleviates the inactivation of the PSII reaction centre.

A guide to Open-JIP, a low-cost open-source chlorophyll fluorometer.

Chlorophyll a fluorescence is the most widely used method to study photosynthesis and plant stress. While several commercial fluorometers are available, there is a need for a low-cost and highly customisable chlorophyll fluorometer. Such a device would aid in performing high-throughput assessment of photosynthesis, as these instruments can be mass-produced. Novel investigations into photosynthesis can also be performed as a result of the user's ability to modify the devices functionality for their specific needs. Motivated by this, we present an open-source chlorophyll fluorometer based on the Kautsky induction curve (OJIP). The instrument consists of low-cost, easy-to-acquire electrical components and an open-source microcontroller (Arduino Mega) whose performance is equivalent to that of commercial instruments. Two 3D printable Open-JIP configurations are presented, one for higher plants and the other for microalgae cells in suspension. Directions for its construction are presented and the instrument is benchmarked against widely used commercial chlorophyll fluorometers.

Mechanism of Photodamage of the Oxygen Evolving Mn Cluster of Photosystem II by Excessive Light Energy.

Photodamage to Photosystem II (PSII) has been attributed either to excessive excitation of photosynthetic pigments or by direct of light absorption by Mn4CaO5 cluster. Here we investigated the time course of PSII photodamage and release of Mn in PSII-enriched membranes under high light illumination at 460 nm and 660 nm. We found that the loss of PSII activity, assayed by chlorophyll fluorescence, is faster than release of Mn from the Mn4CaO5 cluster, assayed by EPR. Loss of PSII activity and Mn release was slower during illumination in the presence of exogenous electron acceptors. Recovery of PSII activity was observed, after 30 min of addition of electron donor post illumination. The same behavior was observed under 460 and 660 nm illumination, suggesting stronger correlation between excessive excitation and photodamage compared to direct light absorption by the cluster. A unified model of PSII photodamage that takes into account present and previous literature reports is presented.

Relative functional and optical absorption cross-sections of PSII and other photosynthetic parameters monitored in situ, at a distance with a time resolution of a few seconds, using a prototype light induced fluorescence transient (LIFT) device.

The prototype light-induced fluorescence transient (LIFT) instrument provides continuous, minimally intrusive, high time resolution (~2s) assessment of photosynthetic performance in terrestrial plants from up to 2m. It induces a chlorophyll fluorescence transient by a series of short flashes in a saturation sequence (180 ~1µs flashlets in <380µs) to achieve near-full reduction of the primary acceptor QA, followed by a relaxation sequence (RQA; 90 flashlets at exponentially increasing intervals over ~30ms) to observe kinetics of QA re-oxidation. When fitted by the fast repetition rate (FRR) model (Kolber et al. 1998) the QA flash of LIFT/FRR gives smaller values for FmQA from dark adapted leaves than FmPAM from pulse amplitude modulated (PAM) assays. The ratio FmQA/FmPAM resembles the ratio of fluorescence yield at the J/P phases of the classical O-J-I-P transient and we conclude that the difference simply is due to the levels of PQ pool reduction induced by the two techniques. In a strong PAM-analogous WL pulse in the dark monitored by the QA flash of LIFT/FRR φPSIIWL ≈ φPSIIPAM. The QA flash also tracks PQ pool reduction as well as the associated responses of ETR QA → PQ and PQ → PSI, the relative functional (σPSII) and optical absorption (aPSII) cross-sections of PSII in situ with a time resolution of ~2s as they relax after the pulse. It is impractical to deliver strong WL pulses at a distance in the field but a longer PQ flash from LIFT/FRR also achieves full reduction of PQ pool and delivers φPSIIPQ ≈ φPSIIPAM to obtain PAM-equivalent estimates of ETR and NPQ at a distance. In situ values of σPSII and aPSII from the QA flash with smaller antenna barley (chlorina-f2) and Arabidopsis mutants (asLhcb2-12, ch1-3 Lhcb5) are proportionally similar to those previously reported from in vitro assays. These direct measurements are further validated by changes in antenna size in response to growth irradiance. We illustrate how the QA flash facilitates our understanding of photosynthetic regulation during sun flecks in natural environments at a distance, with a time resolution of a few seconds.

Photodamage to the oxygen evolving complex of photosystem II by visible light.

Light damages photosynthetic machinery, primarily photosystem II (PSII), and it results in photoinhibition. A new photodamage model, the two-step photodamage model, suggests that photodamage to PSII initially occurs at the oxygen evolving complex (OEC) by light energy absorbed by manganese and that the PSII reaction center is subsequently damaged by light energy absorbed by photosynthetic pigments due to the limitation of electrons to the PSII reaction center. However, it is still uncertain whether this model is applicable to photodamage to PSII under visible light as manganese absorbs visible light only weakly. In the present study, we identified the initial site of photodamage to PSII upon illumination of visible light using PSII membrane fragments isolated from spinach leaves. When PSII samples were exposed to visible light in the presence of an exogenous electron acceptor, both PSII total activity and the PSII reaction centre activity declined due to photodamage. The supplemental addition of an electron donor to the PSII reaction centre alleviated the decline of the reaction centre activity but not the PSII total activity upon the light exposure. Our results demonstrate that visible light damages OEC prior to photodamage to the PSII reaction center, consistent with two-step photodamage model.

The action spectrum of Photosystem II photoinactivation in visible light.

Photosynthesis is always accompanied by light induced damage to the Photosystem II (PSII) which is compensated by its subsequent repair. Photoinhibition of PSII is a complex process, balancing between photoinactivation, protective and repair mechanisms. Current understanding of photoinactivation is limited with competing hypotheses where the photosensitiser is either photosynthetic pigments or the Mn4CaO5 cluster itself, with little consensus on the mechanisms and consequences of PSII photoinactivation. The mechanism of photoinactivation should be reflected in the action spectrum of PSII photoinactivation, but there is a great diversity of the action spectra reported thus far. The only consensus is that PSII photoinactivation is greatest in the UV region of the electromagnetic spectrum. In this review, the authors revisit the methods, technical constraints and the different action spectra of PSII photoinactivation reported to date and compare them against the diverse mechanisms proposed. Upon critical examination of the reported action spectra, a hybrid mechanism of photoinactivation, sensitised by both photosynthetic pigments and the Mn4CaO5 appears to be the most plausible rationalisation.