Biology subjects

Barkan, A.

Publications and source records attributed to Barkan, A..

10 recordsLinked to original sources

bioRxiv2024

Opposing action of photosystem II assembly factors RBD1 and HCF136 underlies light-regulated psbA translation in plant chloroplasts

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. In plants, D1 photodamage activates translation of the chloroplast psbA mRNA encoding D1, providing D1 for PSII repair. Genetic data have implicated three D1 assembly factors in the regulatory mechanism: HCF244 and RBD1 activate psbA translation whereas HCF136 represses psbA translation in the dark. To clarify the regulatory circuit, we analyzed psbA ribosome occupancy in dark-adapted and illuminated rbd1 and hcf136;rbd1 double mutants in Arabidopsis, and in Zm-hcf244 and Zm-hcf136;Zm-hcf244 double mutants in maize. The results show that RBD1 is required for the light-induced recruitment of ribosomes to psbA mRNA but has little effect on psbA ribosome occupancy in the dark. Furthermore, RBD1 is dispensable for psbA translation when HCF136 is absent, indicating that RBD1 activates psbA translation indirectly, by inhibiting HCF136s repressive effect. By contrast, HCF244 is required to recruit ribosomes to psbA mRNA in light, dark, and in the absence of HCF136. These results demonstrate that the step in D1 assembly that is mediated by RBD1 is central to the perception of the D1 photodamage that triggers D1 synthesis, that RBD1 activates psbA translation in the light by relieving the repressive effect of an HCF136-dependent assembly intermediate, and that HCF244 acts independently of these events to activate psbA translation. The results implicate a feature of nascent D1 that is affected by both HCF136 and RBD1 as the signal that reports D1 photodamage to regulate psbA translation rate as needed for PSII repair. Significance StatementThe D1 subunit of photosystem II (PSII) is damaged by light and must be replaced with nascent D1 to maintain photosynthesis. In plants, D1 photodamage triggers D1 synthesis by activating translation of chloroplast psbA mRNA encoding D1. Our results demonstrate that an antagonistic interplay between two photosystem II assembly factors, HCF136 and RBD1, is at the core of the signal transduction process that senses light-induced D1 damage to regulate psbA translation. These findings implicate a feature of nascent D1 that is affected by both HCF136 and RBD1 as the signal that reports D1 photodamage to tune psbA translation as needed for PSII repair. The results elucidate an assembly-coupled translational rheostat that maintains photosynthesis in the face of PSII photodamage.

plant biology↗

bioRxiv2024

Light activates psbA translation in plants by relieving inhibition of translation factor HCF173 by the psbA ORF in cis

The D1 reaction center protein of photosystem II is subject to photooxidative damage. Photodamaged D1 must be replaced with newly synthesized D1 to maintain photosynthesis. In plant chloroplasts, D1 synthesis is coupled to D1 photodamage via regulated translation initiation on psbA mRNA, which encodes D1. Mechanisms underlying this coupling are unclear. We show by analysis of reporter constructs in tobacco that the psbA translational activators HCF173 and HCF244 activate via cis-elements in the psbA UTRs and that the 5 UTR sequence bound by HCF173 is essential for psbA expression. However, the psbA UTRs are not sufficient to program light-regulated translation. Instead, the psbA open reading frame acts in cis to repress translation initiation, and D1 photodamage relieves this repression. A truncated HCF173 mutant conditions constitutively high psbA ribosome occupancy in the dark, implicating HCF173 as a mediator of the repressive signal. We propose a model that is informed by structures of the Complex I assembly factor CIA30/NDUFAF1 positing that D1 photodamage relieves a repressive cotranslational interaction between nascent D1 and HCF173s CIA30 domain, and that the D1 assembly factor HCF136 promotes this interaction. These findings elucidate a translational rheostat that maintains photosynthesis in the face of inevitable photosystem II photodamage.

plant biology↗

bioRxiv2024

Localization of Proteins Involved in the Biogenesis of the Photosynthetic Apparatus to Thylakoid Subdomains in Arabidopsis

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants, and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes, and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

plant biology↗

bioRxiv2024

Translational activation by a synthetic PPR protein elucidates control of psbA translation in Arabidopsis chloroplasts

Translation initiation on psbA mRNA in plant chloroplasts scales with light intensity, providing its gene product, D1, to replace photodamaged D1 in Photosystem II. The psbA translational activator HCF173 has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5-untranslated region (5-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173s footprint in the psbA 5-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts. One sentence summaryA synthetic PPR protein substitutes for HCF173, a non-PPR translational activator in chloroplasts, elucidating HCF173 functions and demonstrating the ability of synthetic PPRs to activate translation.

plant biology↗

bioRxiv2024

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Achieving balanced gene expression within synthetic operons requires a spectrum of expression levels. Here we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5 UTR, which harbors a binding site for PPR10, a protein that activates atpH at the post-transcriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5 UTRs in different design contexts. Notably, high GFP expression was not coupled to stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5 UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning reporter gene expression across a wide range, spanning from 0.02% to 25% of the total soluble cellular protein (TSP). These findings highlight the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

bioengineering↗

bioRxiv2022

Correlated retrograde and developmental regulons implicate multiple retrograde signals as coordinators of chloroplast development in maize

Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of four non-photosynthetic maize mutants and interpreted them in the context of transcriptome dynamics during normal leaf development. We analyzed two albino mutants lacking plastid ribosomes and two chlorotic mutants with thylakoid targeting or plastid transcription defects. The [~]2700 differentially expressed genes fall into six major categories based on the polarity and mutant-specificity of the change. These distinct retrograde responses correlate with distinct developmental dynamics, with down-regulated genes expressed later in normal development and up-regulated genes acting early. Photosynthesis genes are down-regulated specifically in the albino mutants, whereas up-regulated genes are enriched for functions in chloroplast biogenesis and cytosolic translation. TOR signaling is elevated in plastid ribosome-deficient mutants and declines in concert with plastid ribosome buildup during leaf development. Our results implicate three plastid signals as integral players during photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal responds to nutrient consumption by developing chloroplasts and represses TOR, which down-regulates cell proliferation genes early in leaf development. One sentence summaryTranscriptomes of non-photosynthetic maize mutants when interpreted in the context of normal developmental dynamics implicate three plastid signals as coordinators of photosynthetic differentiation. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Alice Barkan (abarkan@uoregon.edu)

plant biology↗

bioRxiv2020

Ribosome profiling elucidates the contribution of differential translation to differential gene expression in bundle sheath and mesophyll cells in maize

The efficiencies offered by C4 photosynthesis have motivated efforts to understand its biochemical, genetic and developmental basis. Reactions underlying C4 traits in most C4 plants are partitioned between two cell types, bundle sheath (BS) and mesophyll (M) cells. RNA-seq has been used to catalog differential gene expression in BS and M cells in maize and several other C4 species. However, the contribution of translational control to maintaining the distinct proteomes of BS and M cells has not been addressed. In this study, we used ribosome profiling (ribo-seq) and RNA-seq to describe translatomes, translational efficiencies, and microRNA abundance in BS and M-enriched fractions of maize seedling leaves. A conservative interpretation of our data revealed 182 genes exhibiting cell-type dependent differences in translational efficiency, 31 of which encode proteins with core roles in C4 photosynthesis. Our results suggest that non-AUG start codons are used preferentially in upstream open reading frames of BS cells, revealed mRNA sequence motifs that correlate with cell type-dependent translation, and identified potential translational regulators that are differentially expressed. In addition, our data expand the set of genes known to be differentially expressed in BS and M cells, including genes encoding transcription factors and microRNAs. These data add to the resources for understanding the evolutionary and developmental basis of C4 photosynthesis and for its engineering into C3 crops.

plant biology↗

bioRxiv2020

In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5 end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5 UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

plant biology↗

bioRxiv2020

Light-induced psbA translation in plants is triggered by photosystem II damage via an assembly-linked autoregulatory circuit

The D1 reaction center protein of Photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair, and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark, and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts. Significance StatementPhotosystem II (PSII) harbors the water-splitting activity underlying oxygenic photosynthesis. The PSII reaction center protein D1 is subject to photodamage and must be replaced with nascent D1 to maintain photosynthetic activity. How new D1 synthesis is coordinated with D1 damage has been a long-standing question. Our results clarify the nature of the light-induced signal that activates D1 synthesis for PSII repair in plants, and suggest an autoregulatory mechanism in which degradation of damaged D1 relieves a repressive interaction between D1 and translational activators in a complex that functions in PSII assembly and repair. This proposed mechanism comprises a responsive switch that couples D1 synthesis to need for D1 during PSII biogenesis and repair.

plant biology↗

bioRxiv2018

The dynamic association of SPO11-1 with conformational changes of meiotic axial elements in maize

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation. Author SummaryMeiosis is essential during sexual reproduction to produce haploid gametes. Recombination is the most crucial step during meiotic prophase I. It enables pairing of homologous chromosomes prior to their reductional division and generates new combinations of genetic alleles for transmission to the next generation. Meiotic recombination is initiated by generating DNA double-strand breaks (DSBs) via SPO11, a topoisomerase-related enzyme. The activity, timing and location of this DSB machinery must be controlled precisely, but how this is achieved remains obscure. Here, we show dynamic localization of SPO11-1 on chromatin during meiotic initiation in maize, yet a similar number of SPO11-1 is able to load onto axial elements (AEs), which accompanies a structural change of the AEs of wild-type meiotic chromosomes. Interestingly, loss of SPO11-1 not only affects DSB formation but also impairs structural alterations of AEs, resulting in abnormally long and curly AEs during early meiosis. Our study provides new insights into SPO11-1 localization during recombination initiation and suggests an intimate relationship between DSB formation and AE structural changes.

molecular biology↗