Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

Differential response of the photosynthetic machinery to dehydration in older and younger resurrection plants.

A group of vascular plants called homoiochlorophyllous resurrection plants evolved unique capabilities to protect their photosynthetic machinery against desiccation-induced damage. This study examined whether the ontogenetic status of the resurrection plant Craterostigma pumilum has an impact on how the plant responds to dehydration at the thylakoid membrane level to prepare cells for the desiccated state. Thus, younger plants (<4 months) were compared with their older (>6 months) counterparts. Ultrastructural analysis provided evidence that younger plants suppressed senescence-like programs that are realized in older plants. During dehydration, older plants degrade specific subunits of the photosynthetic apparatus such as the D1 subunit of PSII and subunits of the cytochrome b6f complex. The latter leads to a controlled down-regulation of linear electron transport. In contrast, younger plants increased photoprotective high-energy quenching mechanisms and maintained a high capability to replace damaged D1 subunits. It follows that depending on the ontogenetic state, either more degradation-based or more photoprotective mechanisms are employed during dehydration of Craterostigma pumilum.

Measuring the dynamic response of the thylakoid architecture in plant leaves by electron microscopy.

The performance of the photosynthesis machinery in plants, including light harvesting, electron transport, and protein repair, is controlled by structural changes in the thylakoid membrane system inside the chloroplasts. In particular, the structure of the stacked grana area of thylakoid membranes is highly dynamic, changing in response to different environmental cues such as light intensity. For example, the aqueous thylakoid lumen enclosed by thylakoid membranes in grana has been documented to swell in the presence of light. However, light-induced alteration of the stromal gap in the stacked grana (partition gap) and of the unstacked stroma lamellae has not been well characterized. Light-induced changes in the entire thylakoid membrane system, including the lumen in both stacked and unstacked domains as well as the partition gap, are presented here, and the functional implications are discussed. This structural analysis was made possible by development of a robust semi-automated image analysis method combined with optimized plant tissue fixation techniques for transmission electron microscopy generating quantitative structural results for the analysis of thylakoid ultrastructure. SIGNIFICANCE STATEMENT: A methodical pipeline ranging from optimized leaf tissue preparation for electron microscopy to quantitative image analysis was established. This methodical development was employed to study details of light-induced changes in the plant thylakoid ultrastructure. It was found that the lumen of the entire thylakoid system (stacked and unstacked domains) undergoes light-induced swelling, whereas adjacent membranes on the stroma side in stacked grana thylakoid approach each other.

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Phragmoplast microtubule dynamics - a game of zones.

Plant morphogenesis relies on the accurate positioning of the partition (cell plate) between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which consists of microtubules, actin filaments, membrane compartments and associated proteins. The phragmoplast forms between daughter nuclei during the transition from anaphase to telophase. As cells are commonly larger than the originally formed phragmoplast, the construction of the cell plate requires phragmoplast expansion. This expansion depends on microtubule polymerization at the phragmoplast forefront (leading zone) and loss at the back (lagging zone). Leading and lagging zones sandwich the 'transition' zone. A population of stable microtubules in the transition zone facilitates transport of building materials to the midzone where the cell plate assembly takes place. Whereas microtubules undergo dynamic instability in all zones, the overall balance appears to be shifted towards depolymerization in the lagging zone. Polymerization of microtubules behind the lagging zone has not been reported to date, suggesting that microtubule loss there is irreversible. In this Review, we discuss: (1) the regulation of microtubule dynamics in the phragmoplast zones during expansion; (2) mechanisms of the midzone establishment and initiation of cell plate biogenesis; and (3) signaling in the phragmoplast.

Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum.

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b6 f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production.

Somaclonal variation does not preclude the use of rice transformants for genetic screening.

Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high-throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non-transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T-DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single-nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium-mediated transformation in addition to those caused by tissue culture-induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10-fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.

Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.).

The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O2(·-), H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.