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In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress—induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress—induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.

Hydrogen sulphide (H 2 S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H 2 S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H 2 S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F v /F m ) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H 2 S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H2S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underline the amelioration of photosynthesis and that H 2 S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification.

Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related 1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG 1a and ATG 13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG 1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG 1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG 1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant's nutritional status.

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

We have studied the physiological and genetic responses of Arabidopsis thaliana L. (Arabidopsis) to gold. The root lengths of Arabidopsis seedlings grown on nutrient agar plates containing 100 mg/L gold were reduced by 75%. Oxidized gold was subsequently found in roots and shoots of these plants, but gold nanoparticles (reduced gold) were only observed in the root tissues. We used a microarray-based study to monitor the expression of candidate genes involved in metal uptake and transport in Arabidopsis upon gold exposure. There was up-regulation of genes involved in plant stress response such as glutathione transferases, cytochromes P450, glucosyl transferases and peroxidases. In parallel, our data show the significant down-regulation of a discreet number of genes encoding proteins involved in the transport of copper, cadmium, iron and nickel ions, along with aquaporins, which bind to gold. We used Medicago sativa L. (alfalfa) to study nanoparticle uptake from hydroponic culture using ionic gold as a non-nanoparticle control and concluded that nanoparticles between 5 and 100 nm in diameter are not directly accumulated by plants. Gold nanoparticles were only observed in plants exposed to ionic gold in solution. Together, we believe our results imply that gold is taken up by the plant predominantly as an ionic form, and that plants respond to gold exposure by up-regulating genes for plant stress and down-regulating specific metal transporters to reduce gold uptake.

Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed.

Key message OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. The chloroplast has its own genetic material and genetic system, but it is also regulated by nuclear-encoded genes. However, little is known about nuclear-plastid regulatory mechanisms underlying early chloroplast biogenesis in rice. In this study, we isolated and characterized a mutant, osppr6 , that showed early chloroplast developmental defects leading to albino leaves and seedling death. We found that the osppr6 mutant failed to form thylakoid membranes. Using map-based cloning and complementation tests, we determined that OsPPR6 encoded a new Pentatricopeptide Repeat (PPR) protein localized in plastids. In the osppr6 mutants, mRNA levels of plastidic genes transcribed by the plastid-encoded RNA polymerase decreased, while those of genes transcribed by the nuclear-encoded RNA polymerase increased. Western blot analyses validated these expression results. We further investigated plastidic RNA editing and splicing in the osppr6 mutants and found that the ndhB transcript was mis-edited and the ycf3 transcript was mis-spliced. Therefore, we demonstrate that OsPPR6, a PPR protein, regulates early chloroplast biogenesis and participates in editing of ndhB and splicing of ycf3 transcripts in rice.

This study was conducted in order to determine the effect of priming with γ-aminobutyric acid (GABA) at 0.5 mM on rice (Oryza sativa L.) seed germination under osmotic stress (OS) induced by polyethylene glycol (30 g/L PEG 6000); and salinity stress (S, 150 mM NaCl) and their combination (OS+S). Priming with GABA significantly alleviated the detrimental effects of OS, S and OS+S on seed germination and seedling growth. The photosynthetic system and water relation parameters were improved by GABA under stress. Priming treatment significantly increased the GABA content, sugars, protein, starch and glutathione reductase. GABA priming significantly reduced Na+ concentrations, proline, free radical and malonaldehyde and also significantly increased K+ concentration under the stress condition. Additionally, the activities of antioxidant enzymes, phenolic metabolism-related enzymes, detoxification-related enzymes and their transcription levels were improved by GABA priming under stress. In the GABA primed-plants, salinity stress alone resulted in an obvious increase in the expression level of Calcineurin B-like Protein-interacting protein Kinases (CIPKs) genes such as OsCIPK01, OsCIPK03, OsCIPK08 and OsCIPK15, and osmotic stress alone resulted in obvious increase in the expression of OsCIPK02, OsCIPK07 and OsCIPK09; and OS+S resulted in a significant up-regulation of OsCIPK12 and OsCIPK17. The results showed that salinity, osmotic stresses and their combination induced changes in cell ultra-morphology and cell cycle progression resulting in prolonged cell cycle development duration and inhibitory effects on rice seedlings growth. Hence, our findings suggested that the high tolerance to OS+S is closely associated with the capability of GABA priming to control the reactive oxygen species (ROS) level by inducing antioxidant enzymes, secondary metabolism and their transcription level. This knowledge provides new evidence for better understanding molecular mechanisms of GABA-regulating salinity and osmotic-combined stress tolerance during rice seed germination and development.

Soil and water contamination from heavy metals and metalloids is one of the most discussed and caused adverse effects on food safety and marketability, crop growth due to phytotoxicity, and environmental health of soil organisms. A hydroponic investigation was executed to evaluate the influence of citric acid (CA) on copper (Cu) phytoextraction potential of jute (Corchorus capsularis L.). Three-weeks-old seedlings of C. capsularis were exposed to different Cu concentrations (0, 50, and 100 μM) with or without the application of CA (2 mM) in a nutrient growth medium. The results revealed that exposure of various levels of Cu by 50 and 100 μM significantly (p < 0.05) reduced plant growth, biomass, chlorophyll contents, gaseous exchange attributes, and damaged ultra-structure of chloroplast in C. capsularis seedlings. Furthermore, Cu toxicity also enhanced the production of malondialdehyde (MDA) which indicated the Cu-induced oxidative damage in the leaves of C. capsularis seedlings. Increasing the level of Cu in the nutrient solution significantly increased Cu uptake by the roots and shoots of C. capsularis seedlings. The application of CA into the nutrient medium significantly alleviated Cu phytotoxicity effects on C. capsularis seedlings as seen by plant growth and biomass, chlorophyll contents, gaseous exchange attributes, and ultra-structure of chloroplast. Moreover, CA supplementation also alleviated Cu-induced oxidative stress by reducing the contents of MDA. In addition, application of CA is helpful in increasing phytoremediation potential of the plant by increasing Cu concentration in the roots and shoots of the plants which is manifested by increasing the values of bioaccumulation (BAF) and translocation factors (TF) also. These observations depicted that application of CA could be a useful approach to assist Cu phytoextraction and stress tolerance against Cu in C. capsularis seedlings grown in Cu contaminated sites.

In buried seedlings, chloroplasts are arrested at the etioplast stage, but they rapidly mature upon emergence of the seedling. Etioplast-chloroplast differentiation is halted through the integration of soil-induced signals, including pressure and the absence of light, although the details on how this information converges to regulate cellular decisions remain unclear. Here, we identify an interdependent transcription module that integrates the mechanical pressure and darkness signals to control chloroplast development in Arabidopsis thaliana. Mutations of ETHYLENE-INSENSITIVE3 (EIN3), the primary transcription factor in the ethylene signaling pathway that is activated in response to mechanical pressure, cause early development of etioplasts in the dark and severe photobleaching upon light exposure. Genetic studies demonstrate that repression of etioplast differentiation by EIN3 requires PHYTOCHROME INTERACTING FACTOR3 (PIF3), a darkness-stabilized bHLH transcription factor. EIN3 and PIF3 directly interact and form an interdependent module to repress the expression of most LIGHT HARVESTING COMPLEX (LHC) genes; overexpressing even one LHC could cause premature development of etioplasts. The EIN3-PIF3 transcription module synergistically halts chloroplast development by interdependently co-occupying the promoters of LHC genes. Thus, our results define a transcriptional regulatory module and provide mechanistic insight on the concerted regulation of chloroplast development by multiple soil-induced signals.