Chloroplast Responses Research Articles

Lipid Signaling through G Proteins.

N-Acylethanolamine (NAE) signaling has received considerable attention in vertebrates as part of the endocannabinoid signaling system, where anandamide acts as a ligand for G protein-coupled cannabinoid receptors. Recent studies indicate that G proteins also are required for some types of NAE signaling in plants. The genetic ablation of the Gβγ dimer or loss of the full set of extra-large G proteins strongly attenuated NAE-induced chloroplast responses in seedlings. Intriguing parallels and distinct differences have emerged between plants and animals in NAE signaling, despite the conserved use of these lipid mediators to modulate cellular processes. Here we compare similarities and differences and identify open questions in a fundamental lipid signaling pathway in eukaryotes with components that are both conserved and diverged in plants.

Integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses

In this study, we performed an integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses by label-free based quantitative proteomic approach. Both salt and osmotic stresses significantly increased the levels of abscisic acid and methyl jasmonate and led to damages of chloroplast ultrastructure. Main parameters of chlorophyll fluorescence and gas exchange showed a significant decline under both stresses. Quantitative proteomic analysis identified 194 and 169 chloroplast-localized differentially accumulated proteins (DAPs) responsive to salt and osmotic stresses, respectively. The abundance of main DAPs involved in light-dependent reaction were increased under salt stress, but decreased in response to osmotic stress. On the contrary, salt stress induced a significant upregulation of the DAPs associated with Calvin cycle, transcription and translation, amino acid metabolism, carbon and nitrogen metabolism, and some of them exhibited a downregulation under osmotic stress. In particular, both treatments significantly upregulated the DAPs involved in plastoglobule development, protein folding and proteolysis, hormone and vitamin synthesis. Finally, we proposed a putative synergistic responsive network of wheat chloroplast proteome under salt and osmotic stresses, aiming to provide new insights into the underlying response and defense mechanisms of wheat chloroplast proteome in response to abiotic stresses. SignificanceSalt and osmotic stresses are the two most common abiotic stresses that severely affect crop growth and productivity. As the main site of photosynthesis of plant cells, the chloroplast also plays important role in plant tolerance to abiotic stress. However, the response of chloroplast proteome to salt and osmotic is still poorly understood by using the traditional two-dimensional electrophoresis (2-DE) method due to a poor resolution of chloroplast protein separation and low throughput identification of differentially accumulated proteins (DAPs). In this study, we employed label-free based quantitative proteomic approach to perform an integrated physiological and large-scale chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses, which laid a solid foundation for future studies into the response and defense mechanisms of wheat chloroplast in response to abiotic stresses.

IM30 IDPs form a membrane-protective carpet upon super-complex disassembly

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated with stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins.

Effect of Plasma Membrane Ion Currents on Chlorophyll Fluorescence and Excitation Quenching in Chara Chloroplasts

Illuminated giant cells of characean algae exhibit membrane excitability as well as the spatial patterns of photosynthesis and transmembrane H+ fluxes. The excitation of plasmalemma under these conditions results in the transient degradation of external alkaline and acid zones and inhibits photosynthesis in the alkaline zones. The generation of action potential in the patterned internodes is followed by cell hyperpolarization that peaks in 1 min and lasts up to 15 min. In order to exclude the influence of drifting resting potential on the chloroplast response to plasma membrane excitation, the voltage clamp mode was applied in this work, and chlorophyll fluorescence changes caused by a short depolarizing pulse were monitored. The depolarizing shift of membrane potential under voltage clamp conditions was found to induce a large depression of $$F_{{\text{m}}}^{{{'}}}$$ chlorophyll fluorescence and photosynthetic activity, provided that inward Ca2+ and Cl– currents were triggered and that a steady-state inward H+ flux (or OH– efflux) persisted before the application of an electric stimulus. The depolarization-induced ion currents measured in the alkaline and acidic cell regions under light and in darkness were found to differ significantly. The results are consistent with the notion that the massive inward H+ flow occurring in the alkaline cell regions under illumination is associated with the acidic shift of cytoplasmic pH. Divergent amplitudes of ionic currents in different cell parts can be partially determined by the presence of numerous plasmalemmal invaginations, charasomes specifically localized in the acid zones, as well as by sharp local changes in external pH in acid zones during the perforation of cell wall with a measuring microelectrode.

Seedling Chloroplast Responses Induced by N-Linolenoylethanolamine Require Intact G-Protein Complexes.

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gβγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis (Arabidopsis thaliana) seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

Structural and Functional Heat Stress Responses of Chloroplasts of Arabidopsis thaliana.

Temperature elevations constitute a major threat to plant performance. In recent years, much was learned about the general molecular mode of heat stress reaction of plants. The current research focuses on the integration of the knowledge into more global networks, including the reactions of cellular compartments. For instance, chloroplast function is central for plant growth and survival, and the performance of chloroplasts is tightly linked to the general status of the cell and vice versa. We examined the changes in photosynthesis, chloroplast morphology and proteomic composition posed in Arabidopsis thaliana chloroplasts after a single or repetitive heat stress treatment over a period of two weeks. We observed that the acclimation is potent in the case of repetitive application of heat stress, while a single stress results in lasting alterations. Moreover, the physiological capacity and its adjustment are dependent on the efficiency of the protein translocation process as judged from the analysis of mutants of the two receptor units of the chloroplast translocon, TOC64, and TOC33. In response to repetitive heat stress, plants without TOC33 accumulate Hsp70 proteins and plants without TOC64 have a higher content of proteins involved in thylakoid structure determination when compared to wild-type plants.

Diurnal changes in chloroplast positioning and photosynthetic traits of C4 grass finger millet

ABSTRACT Mesophyll (M) chloroplasts in finger millet are known to aggregate to the bundle sheath side when leaves are constantly irradiated with extremely high-intensity light. This aggregative movement of M chloroplasts is also observed in natural environment, but whether a natural light regime is effective in inducing the response remains unclear. Abscisic acid is reported to trigger not only the aggregative movement but also stomatal closure, but photosynthetic responses accompanying the aggregative movement also remain unknown.We investigated changes in chloroplast positioning and photosynthetic traits under diurnal patterns of light, mimicking the natural light environment. M chloroplasts showed the aggregative movement with increasing light intensity whether it frequently fluctuated or not, and kept their aggregative positions in the midday. With decreasing light intensity, M chloroplasts returned to the random position in the evening. These results suggest that M chloroplasts often rearrange their intracellular positions during the daytime and that the chloroplast aggregative movement can be induced by a natural regime of light. The chloroplast aggregative movement was observed with increasing stomatal conductance, suggesting that stomatal closure is not crucial to trigger the chloroplast response.

Sensitivity and Responses of Chloroplasts to Heat Stress in Plants.

Increased temperatures caused by global warming threaten agricultural production, as warmer conditions can inhibit plant growth and development or even destroy crops in extreme circumstances. Extensive research over the past several decades has revealed that chloroplasts, the photosynthetic organelles of plants, are highly sensitive to heat stress, which affects a variety of photosynthetic processes including chlorophyll biosynthesis, photochemical reactions, electron transport, and CO2 assimilation. Important mechanisms by which plant cells respond to heat stress to protect these photosynthetic organelles have been identified and analyzed. More recent studies have made it clear that chloroplasts play an important role in inducing the expression of nuclear heat-response genes during the heat stress response. In this review, we summarize these important advances in plant-based research and discuss how the sensitivity, responses, and signaling roles of chloroplasts contribute to plant heat sensitivity and tolerance.

How chloroplasts protect themselves from unfolded proteins.

A genetic screen has identified the first signaling component of the unfolded protein response in chloroplasts.

UV-B Induces Chloroplast Movements in a Phototropin-Dependent Manner.

We examined the impact of UV-B irradiation on chloroplast movements in Arabidopsis leaves. Directional chloroplast movements induced by blue light have been described in multiple plant species. In weak light, chloroplasts accumulate at periclinal cell walls to increase light capture. In strong light, chloroplasts exhibit the avoidance response, as they move towards anticlinal walls to protect the photosynthetic apparatus from light-induced damage. In Arabidopsis, chloroplast movements are triggered by phototropins, phot1 and phot2, which are known as blue/UV-A photoreceptors. We found that irradiation with UV-B of 3.3 µmol·m−2·s−1 induced chloroplast accumulation in wild-type plants. UV-B-triggered accumulation was dependent on the presence of phototropins, especially phot1, but not on UVR8 (the canonical UV-B photoreceptor). Irradiation with strong UV-B of 20 µmol·m−2·s−1 did not induce substantial chloroplast relocations in wild-type leaves. However, in the jac1 mutant, which is defective in chloroplast accumulation, strong UV-B elicited chloroplast avoidance. This indicated that UV-B can also activate signaling to the avoidance response. To assess the possibility of indirect effects of UV-B on chloroplast movements, we examined the impact of UV-B on the actin cytoskeleton, which serves as the motile system for chloroplast movements. While irradiation with UV-B of 3.3 µmol·m−2·s−1 did not affect the actin cytoskeleton, strong UV-B disrupted its structure as shown using an Arabidopsis line expressing Lifeact-green fluorescent protein (GFP). In wild-type plants, pretreatment with strong UV-B attenuated chloroplast responses triggered by subsequent blue light irradiation, further indicating that this UV-B intensity also indirectly affects chloroplast movements. Taken together, our results suggest that the effect of UV-B on chloroplast movement is twofold: it directly induces phototropin-mediated movements; however, at higher intensities, it attenuates the movements in a nonspecific manner.

Chloroplast proteins involved in drought stress response in selected cultivars of common bean (Phaseolus vulgaris L.).

One of the major cell organelles, whose functions are affected during drought stress are chloroplasts. In this study, chloroplast proteome under drought was studied in two cultivars of common bean (Phaseolus vulgaris L), Tiber and more sensitive to drought, Starozagorski čern, which were subjected to drought for 6 and 13days. A comparative proteomic analysis with 2D-DIGE was performed on the isolated chloroplast proteins from leaves. Together, 44 proteins with changed abundance between control and stressed plants were identified with LC-MS/MS from both cultivars. The majority of the identified proteins were involved in photosynthetic processes. The results showed a decrease in abundance in different structure components of photosystem I and II, and ATP synthase, which may indicate a suppression of light-dependent reactions by drought stress. Similar proteomic response for both cultivars after 6 and 13days of drought was observed. Proteins with contrasting abundance patterns between the cultivars or proteins specific for only one cultivar, such as ferredoxin-NADP reductase, photosystem II stability/assembly factor HCF136, curvature thylakoid protein 1B, and plastidial membrane protein porin were pointed out as major identified proteins revealing differential abundance between the cultivars. Taken together, our results provide insight into the molecular response of chloroplasts in common bean under drought stress, whereas conclusions about the tolerance mechanisms require further studies.

The thioredoxin (Trx) redox state sensor protein can visualize Trx activities in the light/dark response in chloroplasts

Thiol-based redox regulation via ferredoxin-thioredoxin (Trx) reductase/Trx controls various functions in chloroplasts in response to light/dark changes. Trx is a key factor of this regulatory system, and five Trx subtypes, including 10 isoforms, have been identified as chloroplast-localized forms in Arabidopsis thaliana These subtypes display distinct target selectivity, and, consequently, they form a complicated redox regulation network in chloroplasts. In this study, we developed a FRET-based sensor protein by combining CFP, YFP, and the N-terminal region of CP12, a redox-sensitive regulatory and Trx-targeted protein in chloroplasts. This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we therefore designated this protein "change in redox state of Trx" (CROST). Using CP12 isoforms, we successfully prepared two types of CROST sensors that displayed different affinities for two major chloroplast Trx isoforms (f-type and m-type). These sensor proteins helped unravel the real-time redox dynamics of Trx molecules in chloroplasts during the light/dark transition.

The increase of photosynthetic carbon assimilation as a mechanism of adaptation to low temperature in Lotus japonicus

Low temperature is one of the most important factors affecting plant growth, it causes an stress that directly alters the photosynthetic process and leads to photoinhibition when severe enough. In order to address the photosynthetic acclimation response of Lotus japonicus to cold stress, two ecotypes with contrasting tolerance (MG-1 and MG-20) were studied. Their chloroplast responses were addressed after 7 days under low temperature through different strategies. Proteomic analysis showed changes in photosynthetic and carbon metabolism proteins due to stress, but differentially between ecotypes. In the sensitive MG-1 ecotype acclimation seems to be related to energy dissipation in photosystems, while an increase in photosynthetic carbon assimilation as an electron sink, seems to be preponderant in the tolerant MG-20 ecotype. Chloroplast ROS generation was higher under low temperature conditions only in the MG-1 ecotype. These data are consistent with alterations in the thylakoid membranes in the sensitive ecotype. However, the accumulation of starch granules observed in the tolerant MG-20 ecotype indicates the maintenance of sugar metabolism under cold conditions. Altogether, our data suggest that different acclimation strategies and contrasting chloroplast redox imbalance could account for the differential cold stress response of both L. japonicus ecotypes.

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics.

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC–MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV–host interaction that might contribute to the symptom development.

Dynamic interaction between Toc159 and blue light receptors at the chloroplast outer envelope of Pisum sativum (L.)

Physiological significance of Pisum sativum TOC GTPases protein phosphoryation has linked the regulation of protein import into chloroplast with the environmental changes. Phototropin 2 is localized at outer membrane of chloroplast. It was crucial to study the relation between Phot2 localization at chloroplast membrane and phosphorylation of TOC GTPases under blue light intensities. Kinase activity of Phot2 towards TOC GTPases was characterized in outer membrane of Pisum sativum. TOC GTPases were specifically and differentially phosphorylated under diffrent light quality and intensities. Gene chip analysis was performed to correlate between expression of TOC complex subunits and blue light receptors genes using A. thaliana. It was found that transcript level of AtToc33 was the highest observed expression with AtToc159 and AtToc75-III. Also, 26 phototropins and photropin-like genes were identified. Coexpression gene netweork of AtPhot2 has revealed a close connection with TPR-containing protein (At1g01320) and chloroplast Toc159 (AT4G15810). Co-immunoprecipitaion assay has evidenced that no intercation was detected between AtPhot2-KD and PsToc34. However, AtPhot2-KD was found to interact with AtToc159. Chloroplast outer and inner envelopes purified from A. thaliana knockout line of Phot2 has phosphorylated AtToc33 and AtToc86/159 in vitro. Hereby, Phot2 is not the specific protein kinase for TOC GTPases. Hence, it was suggested that certain signal cascades may directly or indirectly link phosphorylation of TOC GTPases, protein levels of PHOT-LIKE proteins and irradiation conditions. Therefore, TOC GTPases phosphorylation might be an external regulatory signal to regulate preproteins import into chloroplasts in response to light changes or as a signal of chloroplast biogenesis.

Effects of Chilling on the Structure, Function and Development of Chloroplasts.

Chloroplasts are the organelles that perform energy transformation in plants. The normal physiological functions of chloroplasts are essential for plant growth and development. Chilling is a common environmental stress in nature that can directly affect the physiological functions of chloroplasts. First, chilling can change the lipid membrane state and enzyme activities in chloroplasts. Then, the efficiency of photosynthesis declines, and excess reactive oxygen species (ROS) are produced. On one hand, excess ROS can damage the chloroplast lipid membrane; on the other hand, ROS also represent a stress signal that can alter gene expression in both the chloroplast and nucleus to help regenerate damaged proteins, regulate lipid homeostasis, and promote plant adaptation to low temperatures. Furthermore, plants assume abnormal morphology, including chlorosis and growth retardation, with some even exhibiting severe necrosis under chilling stress. Here, we review the response of chloroplasts to low temperatures and focus on photosynthesis, redox regulation, lipid homeostasis, and chloroplast development to elucidate the processes involved in plant responses and adaptation to chilling stress.

Physiological and Biochemical Responses of Cucumis melo L. Chloroplasts to Low-Phosphate Stress.

Phosphorus (P) is a limiting plant soil nutrient. Long-term low inorganic phosphate (Pi) irreversibly damages plant cells and hinders plant growth. Plants have evolved several adaptive biochemical, physiological, and developmental responses to low-Pi stress. However, little is known about chloroplast responses to low-Pi stress. In this study, we used physiological and biochemical analyses to investigate melon chloroplast responses to low-Pi stress. The results indicated that low-Pi stress impeded melon seedling growth and reduced its dry matter content by inhibiting the photosynthesis. Low-Pi stress reduced the P content in shoots, which inhibited ATP synthase (ATP-ase) activity, and disturbed the proton and electron transport efficiency on chloroplast photosynthetic electron transport chain. In addition, low-Pi stress induced reactive oxygen species (ROS) production in the leaves, which caused membrane peroxidation. Therefore, redox homeostasis was not maintained, and the melon leaves presented with symptoms of photooxidative stress. To mitigate photoinhibition, the melon plants initiated non-photochemical chlorophyll fluorescence quenching (NPQ) initiated by acidification of the thylakoid lumen to dissipate excess excitation energy, significantly improved ROS-scavenging enzyme activity. Based on these experimental results, we concluded that low Pi inhibited photosystem activity and caused photooxidative stress and photoinhibition. To alleviate these negative effects, the plant activated its NPQ mechanism, alternative electron transport pathways, and antioxidant system to protect its chloroplasts.

Adaptive responses of mature giant chloroplasts in the deep-shade lycopod Selaginella erythropus to prolonged light and dark periods.

Deep-shade plants have adapted to low-light conditions by varying morphology and physiology of cells and chloroplasts, but it still remains unclear, if prolonged periods of high-light or darkness induce additional modifications in chloroplasts' anatomy and pigment patterns. We studied giant chloroplasts (bizonoplasts) of the deep-shade lycopod Selaginella erythropus in epidermal cells of mature fully developed microphylls and subjected them to prolonged darkness and high-light conditions. Chloroplast size and ultrastructure were investigated by light and electron microscopy. Physiological traits were studied by pigment analyses, photosynthetic performance of photosystem II, and formation of reactive oxygen species. Results show that (a) thylakoid patterns and shape of mature bizonoplasts vary in response to light and dark conditions. (b) Prolonged darkness induces transitory formation of prolamellar bodies, which so far have not been described in mature chloroplasts. (c) Photosynthetic activity is linked to structural responses of chloroplasts. (d) Photosystem II is less active in the upper zone of bizonoplasts and more efficient in the grana region. (e) Formation of reactive oxygen species reflects the stress level caused by high-light. We conclude that during prolonged darkness, chlorophyll persists and even increases; prolamellar bodies form de novo in mature chloroplasts; bizonoplasts have spatial heterogeneity of photosynthetic performance.

Corrigendum: Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation

To understand why most eukaryotic microalgae accumulate lipids during nitrogen starvation stress, a gene, MiglnB, encoding PII, a signal transduction protein, was cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl. Similarly to its homologues, MiPII contains three conserved T-, B-, and C-loops. In the presence of abundant Mg2+, ATP, and Gln, MiPII upregulates Arg biosynthesis by interacting with the rate-limiting enzyme, MiNAGK, as evidenced by yeast two-hybrid, co-immunoprecipitation assays, and kinetics analysis of enzyme-catalyzed reactions. However, this interaction of MiPII with MiNAGK is reversed by addition of 2-oxoglutarate (2-OG). Moreover, this interaction is present in the chloroplasts of M. incisa, as illustrated cytologically by both immunoelectron microscopy and agroinfiltration of Nicotiana benthamiana leaves to determine the subcellular localization of MiPII with MiNAGK. During the process of nitrogen starvation, soluble Arg levels in M. incisa are modulated by a change in MiNAGK enzymatic activity, both of which are significantly correlated (r = 0.854). A model for the manipulation of Arg biosynthesis via MiPII in M. incisa chloroplasts in response to nitrogen starvation is proposed. The ATP and 2-OG saved from Arg biosynthesis is thus suggested to facilitate the accumulation of fatty acids and triacylglycerol in M. incisa during exposure to nitrogen starvation.