Uncovering the photosystem I assembly pathway in land plants.

Photosystem I (PSI) is one of two large pigment-protein complexes responsible for converting solar energy into chemical energy in all oxygenic photosynthetic organisms. The PSI supercomplex consists of the PSI core complex and peripheral light-harvesting complex I (LHCI) in eukaryotic photosynthetic organisms. However, how the PSI complex assembles in land plants is unknown. Here we describe PHOTOSYSTEM I BIOGENESIS FACTOR 8 (PBF8), a thylakoid-anchored protein in Arabidopsis thaliana that is required for PSI assembly. PBF8 regulates two key consecutive steps in this process, the building of two assembly intermediates comprising eight or nine subunits, by interacting with PSI core subunits. We identified putative PBF8 orthologues in charophytic algae and land plants but not in Cyanobacteria or Chlorophyta. Our data reveal the major PSI assembly pathway in land plants. Our findings suggest that novel assembly mechanisms evolved during plant terrestrialization to regulate PSI assembly, perhaps as a means to cope with terrestrial environments.

Sucrose phosphate synthase 8 is required for the remobilization of carbon reserves in rice stems during grain filling.

Carbon reserve remobilization in stems is closely related to rice grain filling. Sucrose phosphate synthase (SPS) is highly associated with carbon reserve remobilization. In this study, we investigated the expression pattern of SPS genes in various rice tissues, and found that SPS8 is the major SPS isoform in rice stems during the grain-filling stage. We then constructed sps8 mutants using the CRISPR/Cas9 system. The SPS activity of the sps8 mutants was markedly reduced in the stems. In addition, the sps8 mutants exhibited significant starch accumulation in stems. 14C-labelling experiments revealed that the remobilization of non-structural carbohydrates from rice stems to grains was impaired in the sps8 mutants. In the sps8 mutants, grain filling was delayed and yield decreased by 15% due to a reduced percentage of ripened grains. RNA sequencing and quantitative PCR analyses indicated that the genes involved in starch synthesis and degradation were up-regulated in the sps8 mutant stems. In addition, the activity of the enzymes involved in starch synthesis and degradation was increased in the sps8 stems. These results demonstrate that SPS8 is required for carbon reserve remobilization from rice stems to grains, and that its absence may enhance 'futile cycles' of starch synthesis and degradation in rice stems.

Chloroplast gene expression: Recent advances and perspectives.

Chloroplasts evolved from an ancient cyanobacterial endosymbiont more than 1.5 billion years ago. During subsequent coevolution with the nuclear genome, the chloroplast genome has remained independent, albeit strongly reduced, with its own transcriptional machinery and distinct features, such as chloroplast-specific innovations in gene expression and complicated post-transcriptional processing. Light activates the expression of chloroplast genes via mechanisms that optimize photosynthesis, minimize photodamage, and prioritize energy investments. Over the past few years, studies have moved from describing phases of chloroplast gene expression to exploring the underlying mechanisms. In this review, we focus on recent advances and emerging principles that govern chloroplast gene expression in land plants. We discuss engineering of pentatricopeptide repeat proteins and its biotechnological effects on chloroplast RNA research; new techniques for characterizing the molecular mechanisms of chloroplast gene expression; and important aspects of chloroplast gene expression for improving crop yield and stress tolerance. We also discuss biological and mechanistic questions that remain to be answered in the future.

An overview of RNA splicing and functioning of splicing factors in land plant chloroplasts.

RNA splicing refers to a process by which introns of a pre-mRNA are excised and the exons at both ends are joined together. Chloroplast introns are inherently self-splicing ribozymes, but over time, they have lost self-splicing ability due to the degeneration of intronic elements. Thus, the splicing of chloroplast introns relies heavily on nuclear-encoded splicing factors, which belong to diverse protein families. Different splicing factors and their shared intron targets are supposed to form ribonucleoprotein particles (RNPs) to facilitate intron splicing. As characterized in a previous review, around 14 chloroplast intron splicing factors were identified until 2010. However, only a few genetic and biochemical evidence has shown that these splicing factors are required for the splicing of one or several introns. The roles of splicing factors are generally believed to facilitate intron folding; however, the precise role of each protein in RNA splicing remains ambiguous. This may be because the precise binding site of most of these splicing factors remains unexplored. In the last decade, several new splicing factors have been identified. Also, several splicing factors were found to bind to specific sequences within introns, which enhanced the understanding of splicing factors. Here, we summarize recent progress on the splicing factors in land plant chloroplasts and discuss their possible roles in chloroplast RNA splicing based on previous studies.

Autophagy targets Hd1 for vacuolar degradation to regulate rice flowering.

Flowering time (heading date) is a critical agronomic trait that determines the yield and regional adaptability of crops. Heading date 1 (Hd1) is a central regulator of photoperiodic flowering in rice (Oryza sativa). However, how the homeostasis of Hd1 protein is achieved is poorly understood. Here, we report that the nuclear autophagy pathway mediates Hd1 degradation in the dark to regulate flowering. Loss of autophagy function results in an accumulation of Hd1 and delays flowering under both short-day and long-day conditions. In the dark, nucleus-localized Hd1 is recognized as a substrate for autophagy and is subjected to vacuolar degradation via the autophagy protein OsATG8. The Hd1-OsATG8 interaction is required for autophagic degradation of Hd1 in the dark. Our study reveals a new mechanism by which Hd1 protein homeostasis is regulated by autophagy to control rice flowering. Our study also indicates that the regulation of flowering by autophagic degradation of Hd1 orthologs may have arisen over the course of mesangiosperm evolution, which would have increased their flexibility and adaptability to the environment by modulating flowering time.

Katanin-Dependent Microtubule Ordering in Association with ABA Is Important for Root Hydrotropism.

Root hydrotropism refers to root directional growth toward soil moisture. Cortical microtubule arrays are essential for determining the growth axis of the elongating cells in plants. However, the role of microtubule reorganization in root hydrotropism remains elusive. Here, we demonstrate that the well-ordered microtubule arrays and the microtubule-severing protein KATANIN (KTN) play important roles in regulating root hydrotropism in Arabidopsis. We found that the root hydrotropic bending of the ktn1 mutant was severely attenuated but not root gravitropism. After hydrostimulation, cortical microtubule arrays in cells of the elongation zone of wild-type (WT) Col-0 roots were reoriented from transverse into an oblique array along the axis of cell elongation, whereas the microtubule arrays in the ktn1 mutant remained in disorder. Moreover, we revealed that abscisic acid (ABA) signaling enhanced the root hydrotropism of WT and partially rescued the oryzalin (a microtubule destabilizer) alterative root hydrotropism of WT but not ktn1 mutants. These results suggest that katanin-dependent microtubule ordering is required for root hydrotropism, which might work downstream of ABA signaling pathways for plant roots to search for water.

Non-Photochemical Quenching: From Light Perception to Photoprotective Gene Expression.

Light is essential for photosynthesis but light levels that exceed an organism's assimilation capacity can cause serious damage or even cell death. Plants and microalgae have developed photoprotective mechanisms collectively referred to as non-photochemical quenching to minimize such potential damage. One such mechanism is energy-dependent quenching (qE), which dissipates excess light energy as heat. Over the last 30 years, much has been learned about the molecular mechanism of qE in green algae and plants. However, the steps between light perception and qE represented a gap in our knowledge until the recent identification of light-signaling pathways that function in these processes in the green alga Chlamydomonas reinhardtii. In this review, we summarize the high light and UV-mediated signaling pathways for qE in Chlamydomonas. We discuss key questions remaining about the pathway from light perception to photoprotective gene expression in Chlamydomonas. We detail possible differences between green algae and plants in light-signaling mechanisms for qE and emphasize the importance of research on light-signaling mechanisms for qE in plants.

Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit ß and Chloroplast Development in Arabidopsis.

Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz. RT-quantitative real-time PCR, northern blot, and run-on analyses indicated that the PEP-dependent transcription in the pd1 mutant was dramatically impaired, whereas the nuclear-encoded RNA polymerase-dependent transcription was up-regulated. The yeast two-hybrid assays and coimmunoprecipitation experiments showed that the PD1 protein interacts with PEP core subunit ß (PEP-ß), which has been verified to be essential for chloroplast development. The immunoblot analysis indicated that the accumulation of PEP-ß was barely detected in the pd1 mutant, whereas the accumulation of the other essential components of the PEP complex, such as core subunits α and ß', were not affected in the pd1 mutant. These observations suggested that the PD1 protein is essential for the accumulation of PEP-ß and chloroplast development in Arabidopsis, potentially by direct interaction with PEP-ß.

CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts.

Protein-mediated RNA stabilization plays profound roles in chloroplast gene expression. Genetic studies have indicated that chloroplast ndhA transcripts, encoding a key subunit of the NADH dehydrogenase-like complex that mediates photosystem I cyclic electron transport and facilitates chlororespiration, are stabilized by PPR53 and its orthologs, but the underlying mechanisms are unclear. Here, we report that CHLOROPLAST RNA SPLICING 2 (CRS2)-ASSOCIATED FACTOR (CAF) proteins activate SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an ortholog of PPR53 in Arabidopsis thaliana, enhancing their affinity for the 5' ends of ndhA transcripts to stabilize these molecules while inhibiting the RNA endonuclease activity of the SOT1 C-terminal SMR domain. In addition, we established that SOT1 improves the splicing efficiency of ndhA by facilitating the association of CAF2 with the ndhA intron, which may be due to the SOT1-mediated stability of the ndhA transcripts. Our findings shed light on the importance of PPR protein interaction partners in moderating RNA metabolism.

Analysis of the changes of electron transfer and heterogeneity of photosystem II in Deg1-reduced Arabidopsis plants.

Deg1 protease functions in protease and chaperone of PSII complex components, but few works were performed to study the effects of Deg1 on electron transport activities on the donor and acceptor side of PSII and its correlation with the photoprotection of PSII during photoinhibition. Therefore, we performed systematic and comprehensive investigations of electron transfers on the donor and acceptor sides of photosystem II (PSII) in the Deg1-reduced transgenic lines deg1-2 and deg1-4. Both the maximal quantum efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII) decreased significantly in the transgenic plants. Increases in nonphotochemical quenching (NPQ) and the dissipated energy flux per reaction center (DI0/RC) were also shown in the transgenic plants. Along with the decreased D1, CP47, and CP43 content, these results suggested photoinhibition under growth light conditions in transgenic plants. Decreased Deg1 caused inhibition of electron transfer on the PSII reducing side, leading to a decline in the number of QB-reducing centers and accumulation of QB-nonreducing centers. The Tm of the Q band shifted from 5.7 °C in the wild-type plant to 10.4 °C and 14.2 °C in the deg1-2 and deg1-4 plants, respectively, indicating an increase in the stability of S2QA¯ in transgenic plants. PSIIα in the transgenic plants largely reduced, while PSIIß and PSIIγ increased with the decline in the Deg1 levels in transgenic plants suggesting PSIIα centers gradually converted into PSIIß and PSIIγ centers in the transgenic plants. Besides, the connectivity of PSIIα and PSIIß was downregulated in transgenic plants. Our results reveal that downregulation of Deg1 protein levels induced photoinhibition in transgenic plants, leading to loss of PSII activities on both the donor and acceptor sides in transgenic plants. These results give a new insight into the regulation role of Deg1 in PSII electron transport.

OsSWEET14 cooperates with OsSWEET11 to contribute to grain filling in rice.

The grain-filling process is crucial for cereal crop yields, but how the caryopsis of such plants is supplied with sugars, which are produced by photosynthesis in leaves and then transported long distance, is largely unknown. In rice (Oryza sativa), various SWEET family sucrose transporters are thought to have important roles in grain filling. Here, we report that OsSWEET14 plays a crucial part in this process in rice. ossweet14 knockout mutants did not show any detectable phenotypic differences from the wild type, whereas ossweet14;ossweet11 double-knockout mutants had much more severe phenotypes than ossweet11 single-knockout mutants, including strongly reduced grain weight and yield, reduced grain-filling rate, and increased starch accumulation in the pericarp. Both OsSWEET14 and OsSWEET11 exhibited distinct spatiotemporal expression patterns between the early stage of caryopsis development and the rapid grain-filling stage. During the rapid grain-filling stage, OsSWEET14 and OsSWEET11 localized to four key sites: vascular parenchyma cells, the nucellar projection, the nucellar epidermis, and cross cells. These results demonstrate that OsSWEET14 plays an important role in grain filling, and they suggest that four major apoplasmic pathways supply sucrose to the endosperm during the rapid grain-filling stage via the sucrose effluxers SWEET14 and SWEET11.

F-Type ATP Synthase Assembly Factors Atp11 and Atp12 in Arabidopsis.

Atp11p and Atp12p are members of two chaperone families essential for assembly of the mitochondrial ATP synthase in Saccharomyces cerevisiae and Homo sapiens. However, the role of their homologs in higher plants is unclear with regard to the assembly of both chloroplast ATP synthase (cpATPase) and mitochondrial ATP synthase (mtATPase). Here, we show that loss of either Atp11 or Atp12 is lethal in Arabidopsis. While Atp12 is only localized in mitochondria, Atp11 is present both in chloroplasts and mitochondria. Yeast two-hybrid analyses showed that, as their homologs in yeast, Atp11 specifically interacts with the ß subunit of the mtATPase and cpATPase, and Atp12 interacts with the α subunit of the mtATPase, implying that Atp11 and Atp12 fulfill a conserved task during assembly of ATP synthase. However, the binding sites for Atp11 in the ß subunit of mtATPase and cpATPase are slightly different, suggesting that the mechanisms of action may have evolved in different ways. Although Atp11 interacts with cpATPase ß subunit as the two assembly factors BFA3 and BFA1, they bind to different sites of the ß subunit. These results indicate that Atp11 is involved in the assembly of both cpATPase and mtATPase but Atp12 is specifically required for the assembly of mtATPase in higher plants.

Genome-wide transcriptome and proteome profiles indicate an active role of alternative splicing during de-etiolation of maize seedlings.

MAIN CONCLUSION: AS events affect genes encoding protein domain composition and make the single gene produce more proteins with a certain number of genes to satisfy the establishment of photosynthesis during de-etiolation. The drastic switch from skotomorphogenic to photomorphogenic development is an excellent system to elucidate rapid developmental responses to environmental stimuli in plants. To decipher the effects of different light wavelengths on de-etiolation, we illuminated etiolated maize seedlings with blue, red, blue-red mixed and white light, respectively. We found that blue light alone has the strongest effect on photomorphogenesis and that this effect can be attributed to the higher number and expression levels of photosynthesis and chlorosynthesis proteins. Deep sequencing-based transcriptome analysis revealed gene expression changes under different light treatments and a genome-wide alteration in alternative splicing (AS) profiles. We discovered 41,188 novel transcript isoforms for annotated genes, which increases the percentage of multi-exon genes with AS to 63% in maize. We provide peptide support for all defined types of AS, especially retained introns. Further in silico prediction revealed that 58.2% of retained introns have changes in domains compared with their most similar annotated protein isoform. This suggests that AS acts as a protein function switch allowing rapid light response through the addition or removal of functional domains. The richness of novel transcripts and protein isoforms also demonstrates the potential and importance of integrating proteomics into genome annotation in maize.

Comparative analysis reveals gravity is involved in the MIZ1-regulated root hydrotropism.

Hydrotropism is the directed growth of roots toward the water found in the soil. However, mechanisms governing interactions between hydrotropism and gravitropism remain largely unclear. In this study, we found that an air system and an agar-sorbitol system induced only oblique water-potential gradients; an agar-glycerol system induced only vertical water-potential gradients; and a sand system established both oblique and vertical water-potential gradients. We employed obliquely oriented and vertically oriented experimental systems to study hydrotropism in Arabidopsis and tomato plants. Comparative analyses using different hydrotropic systems showed that gravity hindered the ability of roots to search for obliquely oriented water, whilst facilitating roots' search for vertically oriented water. We found that the gravitropism-deficient mutant aux1 showed enhanced hydrotropism in the oblique orientation but impaired root elongation towards water in the vertical orientation. The miz1 mutant exhibited deficient hydrotropism in the oblique orientation but normal root elongation towards water in the vertical orientation. Importantly, in contrast to miz1, the miz1/aux1 double mutant exhibited hydrotropic bending in the oblique orientation and attenuated root elongation towards water in the vertical orientation. Our results suggest that gravitropism is required for MIZ1-regulated root hydrotropism in both the oblique orientation and the vertical orientation, providing further insight into the role of gravity in root hydrotropism.

The Role of Chloroplast Gene Expression in Plant Responses to Environmental Stress.

Chloroplasts are plant organelles that carry out photosynthesis, produce various metabolites, and sense changes in the external environment. Given their endosymbiotic origin, chloroplasts have retained independent genomes and gene-expression machinery. Most genes from the prokaryotic ancestors of chloroplasts were transferred into the nucleus over the course of evolution. However, the importance of chloroplast gene expression in environmental stress responses have recently become more apparent. Here, we discuss the emerging roles of the distinct chloroplast gene expression processes in plant responses to environmental stresses. For example, the transcription and translation of psbA play an important role in high-light stress responses. A better understanding of the connection between chloroplast gene expression and environmental stress responses is crucial for breeding stress-tolerant crops better able to cope with the rapidly changing environment.

An innovative artificial photosystem II constructed from PSII core of Thermosynechococcus vulcanus and LHCII of Pisum sativum - A new approach for studying the function of photosynthetic antenna.

In photosynthesis, the antenna system captures solar energy and transfers the excitations to photosystem II (PSII) core complex where charge separation, water splitting and oxygen evolution occur. In the evolution of photosynthesis from aquatic to terrestrial environments, the structure of PSII core complex was highly conserved while a variety of antenna forms became differentiated. In order to study the principles for energy transport from antenna to the PSII reaction center, we have explored whether the major light harvesting complex of PSII (LHCII) of higher plants can transfer energy to the cyanobacteria PSII core complexes (CC). For this purpose, LHCII from pea and CC from Thermosynechococcus vulcanus were isolated and co-reconstituted into liposome at LHCII:CC molar ratios of 2:1, 4:1 and 6:1, respectively. Chemical-cross linking followed by LC-MS/MS analysis confirmed the biochemical interaction between LHCII and CC in the liposome membrane. The analyses of 77 K fluorescence emission spectra and antenna cross section of PSII indicated that LHCII can transfer energy directly to the cyanobacterial CC. The study has laid the basis for further research on the mechanism of energy transfer from LHCII to PSII CC. This result may also open a new possibility for design and development of new artificial PSII in the application of solar energy conversion.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions.

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding. The root primordia form in the source tissue, and during emergence the adventitious roots penetrate the inner cell layers and the epidermis; however, the mechanisms underlying this emergence remain largely unexplored. Here, we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), the MAP kinases MPK3 and MPK6, and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots (J-ARs) from the root-hypocotyl junctions in Arabidopsis thaliana. ABI4 negatively regulates J-AR emergence, preventing the accumulation of reactive oxygen species and death of epidermal cells, which would otherwise facilitate J-AR emergence. Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4. MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence. We propose that this "double-check" mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.

Pentatricopeptide repeat protein PHOTOSYSTEM I BIOGENESIS FACTOR2 is required for splicing of ycf3.

To gain a better understanding of the molecular mechanisms of photosystem I (PSI) biogenesis, we characterized the Arabidopsis thaliana photosystem I biogenesis factor 2 (pbf2) mutant, which lacks PSI complex. PBF2 encodes a P-class pentatricopeptide repeat (PPR) protein. In the pbf2 mutants, we observed a striking decrease in the transcript level of only one gene, the chloroplast gene ycf3, which is essential for PSI assembly. Further analysis of ycf3 transcripts showed that PBF2 is specifically required for the splicing of ycf3 intron 1. Computational prediction of binding sequences and electrophoretic mobility shift assays reveal that PBF2 specifically binds to a sequence in ycf3 intron 1. Moreover, we found that PBF2 interacted with two general factors for group II intron splicing CHLOROPLAST RNA SPLICING2-ASSOCIATED FACTOR1 (CAF1) and CAF2, and facilitated the association of these two factors with ycf3 intron 1. Our results suggest that PBF2 is specifically required for the splicing of ycf3 intron 1 through cooperating with CAF1 and CAF2. Our results also suggest that additional proteins are required to contribute to the specificity of CAF-dependent group II intron splicing.

Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting.

In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.