Chloroplast Stroma Research Articles

Towards improved dynamic photosynthesis in C3 crops by utilizing natural genetic variation.

Under field environments, fluctuating light conditions induce dynamic photosynthesis, which affects carbon gain by crop plants. Elucidating the natural genetic variations among untapped germplasm resources and their underlying mechanisms can provide an effective strategy to improve dynamic photosynthesis and, ultimately, improve crop yields through molecular breeding approaches. In this review, we first overview two processes affecting dynamic photosynthesis, namely (i) biochemical processes associated with CO2 fixation and photoprotection and (ii) gas diffusion processes from the atmosphere to the chloroplast stroma. Next, we review the intra- and interspecific variations in dynamic photosynthesis in relation to each of these two processes. It is suggested that plant adaptations to different hydrological environments underlie natural genetic variation explained by gas diffusion through stomata. This emphasizes the importance of the coordination of photosynthetic and stomatal dynamics to optimize the balance between carbon gain and water use efficiency under field environments. Finally, we discuss future challenges in improving dynamic photosynthesis by utilizing natural genetic variation. The forward genetic approach supported by high-throughput phenotyping should be introduced to evaluate the effects of genetic and environmental factors and their interactions on the natural variation in dynamic photosynthesis.

Stromal Protein Chloroplast Development and Biogenesis1 Is Essential for Chloroplast Development and Biogenesis in Arabidopsis thaliana.

Although numerous studies have been carried out on chloroplast development and biogenesis, the underlying regulatory mechanisms are still largely elusive. Here, we characterized a chloroplast stromal protein Chloroplast Development and Biogenesis1 (CDB1). The knockout cdb1 mutant exhibits a seedling-lethal and ivory leaf phenotype. Immunoblot and RNA blot analyses show that accumulation of chloroplast ribosomes is compromised in cdb1, resulting in an almost complete loss of plastid-encoded proteins including the core subunits of the plastid-encoded RNA polymerase (PEP) RpoB and RpoC2, and therefore in impaired PEP activity. Orthologs of CDB1 are found in green algae and land plants. Moreover, a protein shows high similarity with CDB1, designated as CDB1-Like (CDB1L), is present in angiosperms. Absence of CDB1L results in impaired embryo development. While CDB1 is specifically located in the chloroplast stroma, CDB1L is localized in both chloroplasts and mitochondria in Arabidopsis. Thus, our results demonstrate that CDB1 is indispensable for chloroplast development and biogenesis through its involvement in chloroplast ribosome assembly whereas CDB1L may fulfill a similar function in both mitochondria and chloroplasts.

Role of Histidine 78 of leaf ferredoxin in the interaction with ferredoxin-NADP+ reductase: regulation of pH dependency and negative cooperativity with NADP(H).

In chloroplast stroma, dynamic pH change occurs in response to fluctuating light conditions. We investigated the pH-dependent electron transfer activity between ferredoxin-NADP+ reductase (FNR) and ferredoxin (Fd) isoproteins from maize leaves. By increasing pH (from 5.5 to 8.5), the electron transfer activity from FNR to photosynthetic-type Fd (Fd1) significantly increased while the activity to nonphotosynthetic type Fd (Fd3) decreased, which was mainly due to their differences in the pH dependency of Km for Fd. Mutation of His78 of Fd1 to Val, corresponding amino acid residue in Fd3, lost the pH dependency, indicating a regulatory role of the His78 in the interaction with FNR. We previously showed that the interaction between FNR and Fd was weakened by the allosteric binding of NADP(H) on FNR. His78Val Fd1 mutant largely suppressed this negative cooperativity. These results indicate the involvement of Fd1 His78 in pH dependency and negative cooperativity in the interaction with FNR.

Protein Targeting Into the Thylakoid Membrane Through Different Pathways.

In higher plants, chloroplasts are essential semi-autonomous organelles with complex compartments. As part of these sub-organellar compartments, the sheet-like thylakoid membranes contain abundant light-absorbing chlorophylls bound to the light-harvesting proteins and to some of the reaction center proteins. About half of the thylakoid membrane proteins are encoded by nuclear genes and synthesized in the cytosol as precursors before being imported into the chloroplast. After translocation across the chloroplast envelope by the Toc/Tic system, these proteins are subsequently inserted into or translocated across the thylakoid membranes through distinct pathways. The other half of thylakoid proteins are encoded by the chloroplast genome, synthesized in the stroma and integrated into the thylakoid through a cotranslational process. Much progress has been made in identification and functional characterization of new factors involved in protein targeting into the thylakoids, and new insights into this process have been gained. In this review, we introduce the distinct transport systems mediating the translocation of substrate proteins from chloroplast stroma to the thylakoid membrane, and present the recent advances in the identification of novel components mediating these pathways. Finally, we raise some unanswered questions involved in the targeting of chloroplast proteins into the thylakoid membrane, along with perspectives for future research.

To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana.

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

Autophagic degradation of the chloroplastic 2-phosphoglycolate phosphatase TaPGLP1 in wheat.

TaPGLP1, a chloroplast stromal 2-phosphoglycolate phosphatase of wheat, is an ATG8-interacting protein and undergoes autophagic degradation in starvation-treated wheat mesophyll protoplasts. Selective autophagy in plants has been shown to target diverse cellular cargoes including whole chloroplasts (Chlorophagy) and several chloroplast components (Piecemeal chlorophagy). Most cargoes of selective autophagy are captured by the autophagic machinery through their direct or indirect interactions with the autophagy-essential factor ATG8. Here, we reported a new ATG8-interacting cargo of piecemeal chlorophagy, the wheat photorespiratory 2-phosphoglycolate phosphatase TaPGLP1. The TaPGLP1-mCherry fusions expressed in wheat protoplasts located in the chloroplast stroma. Strikingly, these fusions are translocated into newly formed chloroplast surface protrusions after a long time incubation of protoplasts in a nutrition-free solution. Visualization of co-expressed TaPGLP1-mCherry and the autophagy marker GFP-TaATG8a revealed physical associations of TaPGLP1-mCherry-accumulating chloroplast protrusions with autophagic structures, implying the delivery of TaPGLP1-mCherry fusions from chloroplasts to the autophagic machinery. TaPGLP1-mCherry fusions were also detected in the GFP-TaATG8a-labelled autophagic bodies undergoing degradation in the vacuoles, which suggested the autophagic degradation of TaPGLP1. This autophagic degradation of TaPGLP1 was further demonstrated by the enhanced stability of TaPGLP1-mCherry in protoplasts with impaired autophagy. Expression of TaPGLP1-mCherry in protoplasts stimulated an enhanced autophagy level probably adopted by cells to degrade the over-produced TaPGLP1-mCherry fusions. Results from gene silencing assays showed the requirement of ATG2s and ATG7s in the autophagic degradation of TaPGLP1. Additionally, TaPGLP1 was shown to interact with ATG8 family members. Collectively, our data suggest that autophagy mediates the degradation of the chloroplast stromal protein TaPGLP1 in starvation-treated mesophyll protoplasts.

Chemical Perturbation of Chloroplast Ca2+ Dynamics in Arabidopsis thaliana Suspension Cell Cultures and Seedlings.

Ca2+ signaling is part of universal signal transduction pathways to respond to external and internal stimuli or stress and in plants plays a central role in chloroplasts, such as in the regulation of photosynthetic enzymes or the transition from light to dark. Only recently, the underlying molecular machinery, e.g., transporters and channels that enable chloroplast Ca2+ fluxes, has started to be elucidated. However, chemical tools to specifically perturb these chloroplast Ca2+ fluxes are largely lacking. Here, we describe an efficient aequorin-based system in Arabidopsis thaliana suspension cell cultures to screen for chemicals that alter light-to-dark-induced chloroplast stroma Ca2+ signals. Subsequently, the effect of the hits on chloroplast Ca2+ signals is validated in Arabidopsis seedlings. The research lays a foundation for the identification of novel proteins involved in Ca2+ transport in chloroplast stroma under light-to-dark transition and for investigating the interaction of chloroplast Ca2+ signaling with photosynthesis in general.

Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

CO2-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii.

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO2-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO2-limiting conditions (low-CO2 [LC] and very low-CO2 [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO2, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO2. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO2, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO2. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO2 concentration with ∼7 µM as the boundary.

Identification of New Proteins and Potential Mitochondrial F1F0-ATPase Inhibitor Factor 1-Associated Mechanisms in Arabidopsis thaliana Using iTRAQ-Based Quantitative Proteomic Analysis

The mitochondrial synthesis of ATP makes a vital contribution to the growth and development of biological organisms, in which the enzyme mitochondrial F1F0-ATP synthase plays a pivotal role, in that it can either synthesize or hydrolyze cellular ATP. The finding of our previous study revealed that mitochondrial F1F0-ATPase inhibitor factor 1 (IF1) in Arabidopsis thaliana has a conserved function as an endogenous inhibitor affecting cellular energy status and plays an important role in plant growth and reproduction, particularly in fertility. In this study, to gain an insight into IF1-related traits, we performed isobaric tags for relative and absolute quantitation labeling analysis. In total, 67 of 4778 identified proteins were identified as differentially expressed proteins (DEPs; 59 up-regulated and 8 down-regulated) between wild-type and if1 mutant Arabidopsis thaliana seedlings. Gene ontology enrichment analysis revealed that these DEPs were the most significantly enriched in pathways such as “long-day photoperiodism, flowering,” “positive regulation of protein import into chloroplast stroma,” and “pollen sperm cell differentiation,” which are closely associated with reproductive development. Moreover, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis was the pathway most significantly enriched with DEPs. Collectively, our results revealed a global shift in protein abundance patterns corresponding to AtIF1 mutation, entailing changes in the abundance of multiple key proteins and metabolic processes, which will provide a valuable proteomic foundation for future studies.

The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Indirect Export of Reducing Equivalents From the Chloroplast to Resupply NADP for C3 Photosynthesis-Growing Importance for Stromal NAD(H)?

Plant productivity greatly relies on a flawless concerted function of the two photosystems (PS) in the chloroplast thylakoid membrane. While damage to PSII can be rapidly resolved, PSI repair is complex and time-consuming. A major threat to PSI integrity is acceptor side limitation e.g., through a lack of stromal NADP ready to accept electrons from PSI. This situation can occur when oscillations in growth light and temperature result in a drop of CO2 fixation and concomitant NADPH consumption. Plants have evolved a plethora of pathways at the thylakoid membrane but also in the chloroplast stroma to avoid acceptor side limitation. For instance, reduced ferredoxin can be recycled in cyclic electron flow or reducing equivalents can be indirectly exported from the organelle via the malate valve, a coordinated effort of stromal malate dehydrogenases and envelope membrane transporters. For a long time, the NADP(H) was assumed to be the only nicotinamide adenine dinucleotide coenzyme to participate in diurnal chloroplast metabolism and the export of reductants via this route. However, over the last years several independent studies have indicated an underappreciated role for NAD(H) in illuminated leaf plastids. In part, it explains the existence of the light-independent NAD-specific malate dehydrogenase in the stroma. We review the history of the malate valve and discuss the potential role of stromal NAD(H) for the plant survival under adverse growth conditions as well as the option to utilize the stromal NAD(H) pool to mitigate PSI damage.

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns.

Heterologous synthesis of a biophysical CO2-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C3 plant chloroplast is functional bicarbonate (HCO3−) transporters and vectorial CO2-to-HCO3− converters. Engineering strategies aim to locate these transporters and conversion systems to the C3 chloroplast, enabling elevation of HCO3− concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO3− transporter, and CO2-to-HCO3− converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.

Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Osj10gBTF3-Mediated Import of Chloroplast Protein Is Essential for Pollen Development in Rice.

Chloroplasts are crucial organelles for the generation of fatty acids and starch required for plant development. Nascent polypeptide-associated complex (NAC) proteins have been implicated in development as transcription factors. However, their chaperone roles in chloroplasts and their relationship with pollen development in plants remain to be elucidated. Here, we demonstrated that Osj10gBTF3, a NAC protein, regulates pollen and chloroplast development in rice by coordinating with a Hsp90 family chaperone OsHSP82 to mediate chloroplast import. Knockout of Osj10gBTF3 affects pollen and chloroplast development and significantly reduces the accumulation of fertility-related chloroplast protein OsPPR676. Both Osj10gBTF3 and OsHSP82 interact with OsPPR676. Interestingly, the interaction between OsHSP82 and OsPPR676 is only found in the cytoplasm, while the interaction between Osj10gBTF3 and OsPPR676 also occurs inside the chloroplast. The chloroplast stroma chaperone OsCpn60 can also be co-precipitated with Osj10gBTF3, but not with OsHSP82. Further investigation indicates that Osj10gBTF3 enters the chloroplast stroma possibly through the inner chloroplast membrane channel protein Tic110 and then recruits OsCpn60 for the folding or assembly of OsPPR676. Our results reveal a chaperone role of Osj10gBTF3 in chloroplast import different from Hsp90 and provide a link between chloroplast transport and pollen development in rice.

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H2O2) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H2O2 sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔCR to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H2O2 levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H2O2 gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H2O2 biology in plant cells.

Comparative transcriptome analysis of different nitrogen responses in low-nitrogen sensitive and tolerant maize genotypes

Although previous researches have greatly increased our general knowledge on plant responses to nitrogen (N) stress, a comprehensive understanding of the different responses in crop genotypes is still needed. This study evaluated 304 maize accessions for low-N tolerance under field conditions, and selected the low-N sensitive Ye478 and low-N tolerant Qi319 for further investigations. After a 5-day low-N treatment, the typical N-deficient phenotype with yellowing older leaves was observed in Ye478 but not in Qi319. After the 5-day low-N stress, 16 RNA libraries from leaf and root of Ye478 and Qi319 were generated. The differentially expressed genes (DEGs) in the root of Qi319 up-regulated by special N deficiency were mainly enriched in energy-related metabolic pathways, including tricarboxylic acid metabolic process and nicotinamide metabolic process. Consistent with yellowing older leaves only observed in Ye478, the special N deficiency-responsive DEGs related to thylakoid, chloroplast, photosynthetic membrane, and chloroplast stroma pathways were repressed by low-N stress in Ye478. A total of 216 transcription factors (TFs), including ZmNLP5, were identified as special N deficiency-responsive TFs between Qi319 and Ye478, indicating the importance of transcriptional regulation of N stress-responsive pathway in different tolerance to low-N stress between crop genotypes. In addition, 15 miRNAs were identified as DEGs between Qi319 and Ye478. Taken together, this study contributes to the understanding of the genetic variations and molecular basis of low-N tolerance in maize.

A homolog of GuidedEntry of Tail-anchored proteins3 functions in membrane-specific protein targeting in chloroplasts of Arabidopsis.

The chloroplasts and mitochondria of photosynthetic eukaryotes contain proteins that are closely related to cytosolic Guided Entry of Tail-anchored proteins3 (Get3). Get3 is a targeting factor that efficiently escorts tail-anchored (TA) proteins to the ER. Because other components of the cytosolic-targeting pathway appear to be absent in organelles, previous investigators have asserted that organellar Get3 homologs are unlikely to act as targeting factors. However, we show here both that the Arabidopsis thaliana chloroplast homolog designated as GET3B is structurally similar to cytosolic Get3 proteins and that it selectively binds a thylakoid-localized TA protein. Based on genetic interactions between a get3b mutation and mutations affecting the chloroplast signal recognition particle-targeting pathway, as well as changes in the abundance of photosynthesis-related proteins in mutant plants, we propose that GET3B acts primarily to direct proteins to the thylakoids. Furthermore, through molecular complementation experiments, we show that function of GET3B depends on its ability to hydrolyze ATP, and this is consistent with action as a targeting factor. We propose that GET3B and related organellar Get3 homologs play a role that is analogous to that of cytosolic Get3 proteins, and that GET3B acts as a targeting factor in the chloroplast stroma to deliver TA proteins in a membrane-specific manner.

Toward a comprehensive understanding of global vegetation CO2 assimilation from space.

Large-scale global analysis of the relationship between growing season solar-induced chlorophyll fluorescence (SIF) and gross primary productivity (GPP), indicated by the GPP/SIF ratio, varied greatly with higher values found in wet-and-cold climate regions and lower values found in hot-and-dry climate regions. Such pattern has been shown to be most influenced by the environmental factor of moisture availability.