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The discovery of a nonphotosynthetic plastid in malaria and other apicomplexan parasites has sparked a contentious debate about its evolutionary origin. Molecular data have led to conflicting conclusions supporting either its green algal origin or red algal origin, perhaps in common with the plastid of related dinoflagellates. This distinction is critical to our understanding of apicomplexan evolution and the evolutionary history of endosymbiosis and photosynthesis; however, the two plastids are nearly impossible to compare due to their nonoverlapping information content. Here we describe the complete plastid genome sequences and plastid-associated data from two independent photosynthetic lineages represented by Chromera velia and an undescribed alga CCMP3155 that we show are closely related to apicomplexans. These plastids contain a suite of features retained in either apicomplexan (four plastid membranes, the ribosomal superoperon, conserved gene order) or dinoflagellate plastids (form II Rubisco acquired by horizontal transfer, transcript polyuridylylation, thylakoids stacked in triplets) and encode a full collective complement of their reduced gene sets. Together with whole plastid genome phylogenies, these characteristics provide multiple lines of evidence that the extant plastids of apicomplexans and dinoflagellates were inherited by linear descent from a common red algal endosymbiont. Our phylogenetic analyses also support their close relationship to plastids of heterokont algae, indicating they all derive from the same endosymbiosis. Altogether, these findings support a relatively simple path of linear descent for the evolution of photosynthesis in a large proportion of algae and emphasize plastid loss in several lineages (e.g., ciliates, Cryptosporidium, and Phytophthora).

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Rice (Oryza sativa L.) frequently suffers in late spring from severe damage due to cold spells, which causes the block of chlorophyll biosynthesis during early rice seedling greening. However, the inhibitory mechanism by which this occurs is still unclear. To explore the responsive mechanism of rice seedlings to low temperatures during greening, the effects of chilling stress on chlorophyll biosynthesis and plastid development were studied in rice seedlings. Chlorophyll biosynthesis was obviously inhibited and chlorophyll accumulation declined under low temperatures during greening. The decrease in chlorophyll synthesis was due to the inhibited synthesis of δ-aminolevulinic acid (ALA) and the suppression of conversion from protochlorophyllide (Pchlide) into chlorophylls (Chls). Meanwhile, the activities of glutamate-1-semialdehyde transaminase (GSA-AT), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were downregulated under low temperatures. Further investigations showed that chloroplasts at 18 °C had loose granum lamellae, while the thylakoid and lamellar structures of grana could hardly develop at 12 °C after 48 h of greening. Additionally, photosystem II (PSII) and photosystem I (PSI) proteins obviously declined in the stressed seedlings, to the point that the PSII and PSI proteins could hardly be detected after 48 h of greening at 12 °C. Furthermore, the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) and cell death were all induced by low temperature. Chilling stress had no effect on the development of epidermis cells, but the stomata were smaller under chilling stress than those at 28 °C. Taken together, our study promotes more comprehensive understanding in that chilling could inhibit chlorophyll biosynthesis and cause oxidative damages during greening.

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.

Plastids perform many essential functions in plant metabolism including photosynthesis, synthesis of metabolites, and stress signaling. The most prominent type in green leaves is the chloroplast which contains thylakoids, plastoglobules, and starch. As these structures are closely linked to the metabolism of chloroplasts, changes during plant growth and development and during environmental stress situations are likely to occur. The aim of this study was to characterize changes in size and ultrastructure of chloroplast on cross-sections of leaves during high light stress, Botrytis infection, and dark induced senescence by quantitative transmission electron microscopy (TEM).The size of chloroplasts on cross sections of leaves decreased significantly when plants were subject to high light (49%), Botrytis infection (58%), and senescence (71%). The number of chloroplasts on cross sections of the palisade cell layer and spongy parenchyma, respectively, decreased significantly in plants exposed to high light conditions (48% and 29%), infected with Botrytis (48% and 46%), and during senescence (78% and 80%). Thylakoids on cross-sections of chloroplasts decreased significantly in plants exposed to high light (22%), inoculated with Botrytis cinerea (36%), and senescence (51%). This correlated with a massive increase in plastoglobules on cross-sections of chloroplasts of 88%, 2,306% and 19,617%, respectively. Starch contents on cross sections of chloroplasts were completely diminished in all three stress scenarios. These results demonstrate that the decrease in the number and size of chloroplasts is a reliable stress marker in plants during abiotic and biotic stress situations which can be easily detected with a light microscope. Further, lack of starch, the occurrence of large plastoglobules and decrease in thylakoids can also be regarded as reliable stress marker in plants which can be detected by TEM.

In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice.

Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with piastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from piastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.

The plastid-encoded RNA polymerase serves as the principal transcription machinery within chloroplasts, transcribing over 80% of all primary plastid transcripts. This polymerase consists of a prokaryotic-like core enzyme known as the plastid-encoded RNA polymerase core, and is supplemented by newly evolved associated proteins known as PAPs. However, the architecture of the plastid-encoded RNA polymerase and the possible functions of PAPs remain unknown. Here, we present the cryo-electron microscopy structure of a 19-subunit plastid-encoded RNA polymerase complex derived from spinach ( Spinacia oleracea ). The structure shows that the plastid-encoded RNA polymerase core resembles bacterial RNA polymerase. Twelve PAPs and two additional proteins (FLN2 and pTAC18) bind at the periphery of the plastid-encoded RNA polymerase core, forming extensive interactions that may facilitate complex assembly and stability. PAPs may also protect the complex against oxidative damage and has potential functions in transcriptional regulation. This research offers a structural basis for future investigations into the functions and regulatory mechanisms governing the transcription of plastid genes. The plastid-encoded RNA polymerase serves as the principal transcription machinery within chloroplasts. Here, the authors present the cryo-electron microscopy structure of a 19-subunit plastid-encoded RNA polymerase complex derived from spinach.

Background and AimsThe Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.MethodsIn this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.Key ResultsCryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.ConclusionsThis high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.

Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zeamays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis.