Multiple K+ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K + transporters is limited. Potassium transport and a well-balanced K + homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K + transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higherorder loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na + /H + antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.

Understanding and improving the productivity and robustness of plant photosynthesis requires high-throughput phenotyping under environmental conditions that are relevant to the field. Here we demonstrate the dynamic environmental photosynthesis imager (DEPI), an experimental platform for integrated, continuous, and high-throughput measurements of photosynthetic parameters during plant growth under reproducible yet dynamic environmental conditions. Using parallel imagers obviates the need to move plants or sensors, reducing artifacts and allowing simultaneous measurement on large numbers of plants. As a result, DEPI can reveal phenotypes that are not evident under standard laboratory conditions but emerge under progressively more dynamic illumination. We show examples in mutants of Arabidopsis of such "emergent phenotypes" that are highly transient and heterogeneous, appearing in different leaves under different conditions and depending in complex ways on both environmental conditions and plant developmental age. These emergent phenotypes appear to be caused by a range of phenomena, suggesting that such previously unseen processes are critical for plant responses to dynamic environments.

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H 2 O 2 . We thus tested the hypothesis that CEF can be activated by H 2 O 2 in vivo. CEF was strongly increased by H 2 O 2 both by infiltration or in situ production by chloroplastlocalized glycolate oxidase, implying that H 2 O 2 can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H 2 O 2 , suggesting activation of previously expressed CEF-related machinery. H 2 O 2 -dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H 2 O 2 . We propose that H 2 O 2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants. I n oxygenic photosynthesis, linear electron flow (LEF) is the process by which light energy is captured to drive the extraction of electrons and protons from water and transfer them through a system of electron carriers to reduce NADPH. LEF is coupled to proton translocation into the thylakoid lumen, generating an electrochemical gradient of protons ðΔμ H +Þ or proton motive force (pmf). The pmf drives the synthesis of ATP to power the reactions of the Calvin-Benson-Bassham (CBB) cycle and other essential metabolic processes in the chloroplast. The pmf is also a key regulator of photosynthesis in that it activates the photoprotective q E response to dissipate excess light energy and downregulates electron transfer by controlling the rate of oxidation of plastoquinol at the cytochrome b 6 f complex (b 6 f), thus preventing the buildup of reduced intermediates (1, 2).LEF results in the transfer or deposition into the lumen of three protons for each electron transferred through PSII, plastoquinone (PQ), b 6 f, plastocyanin, and photosystem I (PSI) to ferredoxin (Fd). The synthesis of one ATP is thought to require the passage of 4.67 protons through the ATP synthase, so that LEF should produce a ratio of ATP/NADPH of about 1.33; this ratio is too low to sustain the CBB cycle or supply ATP required for translation, protein transport, or other ATP-dependent processes (3). In addition, the relative demands for ATP and NADPH can change dramatically depending on environmental, developmental, and other factors, leading to rapid energy imbalances that require dynamical regulation of ATP/NADPH balance.Several alternative electron flow pathways in the chloroplast have been proposed to augment ATP production, thus balancing the ATP/NADPH ...

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.DOI: http://dx.doi.org/10.7554/eLife.16921.001

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

In April 2018, rotted ‘Lamoka’ tubers were received from a commercial storage facility (<1% incidence) in St. Joseph County, Michigan by the MSU Potato & Sugar Beet Pathology and Plant & Pest Diagnostics programs. Dense circular colonies of white fungal-like growth were observed on the surface of the tubers, and internal tissues were watery and spongy with gray to brown discoloration (Supplemental Figure 1). Tubers had a strong, sweet alcoholic odor. External and internal tuber tissues were surface disinfested in 0.825% sodium hypochlorite for 1 min, rinsed twice in sterile distilled water, blotted on sterile filter paper, and placed onto 1.5% (w/v) water agar (WA). After 3 days at 21-24°C and ambient light conditions, septate, branched mycelia and hyaline, cylindrical, single-celled conidia 5.2-8.9 µm x 3.6-5.2 µm (n=20 arthrospores) were observed singly or in chains (Supplemental Figure 2A&B). On potato dextrose agar (PDA), colonies were white, circular, and dense (Supplemental Figure 2C). These observations matched morphological descriptions of Geotrichum candidum (Carmichael 1957). No Pythium or Phytophthora spp. were detected. A mono-conidial isolate of the fungus was obtained and maintained on PDA. DNA was extracted from mycelia using a DNeasy plant mini kit (QIAGEN). Fragments of the internal transcribed spacer (ITS) and 18S ribosomal RNA gene regions were amplified using primers ITS1F/4 primers and NS3/8, respectively (White et al. 1990). Purified PCR products (QIAquick PCR purification kit, QIAGEN) were submitted for Sanger sequencing at the Genomics Research Technology Support Facility (East Lansing, MI). The ITS1F/4 and NS3/8 consensus sequences (OP142324 and OP153873) aligned with GenBank accessions of G. candidum KY103456.1 (100% identity) and JF262193.1 (99.75% identity), respectively. Healthy ‘Lamoka’ tubers were rinsed with tap water, surface disinfested in 0.825% sodium hypochlorite for 15 min, rinsed twice in sterile distilled water, and blotted dry on sterile paper towel. Ten tubers were inoculated by placing 10-mm diameter fully colonized agar plugs, excised from the margin of a 9-day-old PDA culture, onto the surface of each tuber (Duellman et al. 2021). Ten tubers were mock-inoculated using sterile PDA. Tubers were placed in a moist chamber and incubated in the dark at room temperature. After nine days, inoculated tubers exhibited white colony growth on tuber surfaces and an alcoholic scent was present. After 27 days, internal tissues were rubbery, but no discoloration was observed. No rubbery rot symptoms were observed on the control tubers. Samples were excised 1 cm laterally from and vertically beneath the inoculation site. Tissues were surface disinfested as described above and plated on 1.5% WA. After 9 days, a Geotrichum sp. identical to the original isolate was confirmed in 50% of samples from inoculated tubers. No Geotrichum sp. were detected from mock-inoculated tubers. Since 2018, G. candidum has been confirmed in commercial storages in three counties in the Lower Peninsula (incidences up to 1-2%). Geotrichum candidum was recently reported causing rubbery rot of potato in Idaho (Duellman et al. 2021); however, to our knowledge this is the first report of rubbery rot in Michigan. Despite increasing detection frequencies, incidences remain low and spread in storage appears limited. Seed decay leading to stand loss (incidence 1-3%) was observed after planting infected lots, which should be avoided or minimized.

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (D) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that D-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.