Lixin Zhang

Summary: R‐loops, three‐stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single‐stranded DNA, play critical roles in gene expression and genome stability. How R‐loop homeostasis is integrated into chloroplast gene expression remains largely unknown. We found an unexpected function of FtsHi1, an inner envelope membrane‐bound AAA‐ATPase in chloroplast R‐loop homeostasis of Arabidopsis thaliana. Previously, this protein was shown to function as a component of the import motor complex for nuclear‐encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP‐dependent helicase that efficiently unwinds both DNA–DNA and DNA–RNA duplexes, thereby preventing R‐loop accumulation. Over‐accumulation of R‐loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear‐ and chloroplast‐encoded subunits of multi‐protein photosynthetic complexes. This study suggests a mechanical link between protein import and R‐loop homeostasis in chloroplasts of higher plants.

An RNA-chaperone-like protein stabilizes 3ʹ processed transcripts of photosynthetic electron transfer B (petB) and stimulates the translation of petD and petA i

The high temperature requirement A (HtrA) proteases (also termed Deg proteases) play important roles in diverse organisms by regulating protein quality and quantity. One of the 16 Arabidopsis homologs, Deg9, is located in the nucleus where it modulates cytokinin- and light-mediated signalling via degrading the ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4). To uncover the structural features underlying the proteolytic activity of Deg9, we determined its crystal structure. Unlike the well-established trimeric building block of HtrAs, Deg9 displays a novel octameric structure consisting of two tetrameric rings that have distinct conformations. Based on the structural architecture, we generated several mutant variants of Deg9, determined their structure and tested their proteolytic activity towards ARR4. The results of the structural and biochemical analyses allowed us to propose a model for a novel mechanism of substrate recognition and activity regulation of Deg9. In this model, protease activation of one tetramer is mediated by en-bloc reorientation of the protease domains to open an entrance for the substrate in the opposite (inactive) tetramer. This study provides the structural basis for understanding how the levels of nuclear signal components are regulated by a plant protease.

Significance: Proper timing of flowering transition is vital for the reproductive success of plants and orchestrated by endogenous and external factors; however, the mechanisms of how plants regulate flowering under high light are not well understood. In this study, we show that promotion of flowering by high light involves the coupling of chloroplast retrograde signals and transcriptional silencing of the floral repressor FLOWERING LOCUS C ( FLC ). In response to high light, a chloroplast envelope-localized transcription factor, PTM, releases its N-terminal fragment through processing to associate with the chromatin remodeler FVE and suppresses FLC transcription. This report describes the molecular basis for a unique intracellular signaling pathway derived from chloroplasts in which plants regulate the developmental timing of the flowering transition.

Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3ω-mediated Ca²⁺-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca²⁺ signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain.

Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components. Significance Selective protein proteolysis is essential for many plant signal transduction pathways and regulates developmental stages of a plant. In addition to the well-characterized ubiquitin-proteasome system, other factors appear to be involved in the degradation of plant signaling components. Here we describe the function of the serine protease degradation of periplasmic protein 9 (DEG9) in plant signaling. We found that DEG9 mediates the degradation of ARABIDOPSIS RESPONSE REGULATOR 4, which is critical for regulating the cross-talk between cytokinin and light-signaling pathways. This study adds to our knowledge about the function of DEG proteases, which are common in the plant kingdom, and emphasizes their importance in plant development.

The chloroplast ATP synthase, a multisubunit complex in the thylakoid membrane, catalyzes the light-driven synthesis of ATP, thereby supplying the energy for carbon fixation during photosynthesis. The chloroplast ATP synthase is composed of both nucleus- and chloroplast-encoded proteins that have required the evolution of novel mechanisms to coordinate the biosynthesis and assembly of chloroplast ATP synthase subunits temporally and spatially. Here we have elucidated the assembly mechanism of the α₃β₃γ core complex of the chloroplast ATP synthase by identification and functional characterization of a key assembly factor, PAB (PROTEIN IN CHLOROPLAST ATPASE BIOGENESIS). PAB directly interacts with the nucleus-encoded γ subunit and functions downstream of chaperonin 60 (Cpn60)-mediated CF₁γ subunit folding to promote its assembly into the catalytic core. PAB does not have any recognizable motifs or domains but is conserved in photosynthetic eukaryotes. It is likely that PAB evolved together with the transfer of chloroplast genes into the nucleus to assist nucleus-encoded CF₁γ assembly into the CF₁ core. Such coordination might represent an evolutionarily conserved mechanism for folding and assembly of nucleus-encoded proteins to ensure proper assembly of multiprotein photosynthetic complexes. Significance The assembly of chloroplast ATP synthase, a multiprotein complex made of subunits of mixed genetic origin, is essential for organelle biogenesis and organism survival. Assembly of multiprotein complexes relies not only on the proper action of universal folding chaperones but also on that of specific assembly chaperones that orchestrate the joining of individual components into higher-order complexes. In this work, we have identified a specific assembly chaperone for chloroplast ATP synthase and shown that it functions downstream of the Cpn60 chaperone to promote assembly of the catalytically active core of the chloroplast ATP synthase. Such collaboration between folding and assembly chaperones appears to be a widely used cellular strategy for the folding and assembly of nucleus-encoded proteins into multiprotein photosynthetic complexes.