Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

The cysteine-rich receptor-like kinase CRK4 contributes to the different drought stress response between Columbia and Landsberg erecta.

The natural variation between Arabidopsis (Arabidopsis thaliana) ecotypes Columbia (Col) and Landsberg erecta (Ler) strongly affects abscisic acid (ABA) signalling and drought tolerance. Here, we report that the cysteine-rich receptor-like protein kinase CRK4 is involved in regulating ABA signalling, which contributes to the differences in drought stress tolerance between Col-0 and Ler-0. Loss-of-function crk4 mutants in the Col-0 background were less drought tolerant than Col-0, whereas overexpressing CRK4 in the Ler-0 background partially to completely restored the drought-sensitive phenotype of Ler-0. F1 plants derived from a cross between the crk4 mutant and Ler-0 showed an ABA-insensitive phenotype with respect to stomatal movement, along with reduced drought tolerance like Ler-0. We demonstrate that CRK4 interacts with the U-box E3 ligase PUB13 and enhances its abundance, thus promoting the degradation of ABA-INSENSITIVE 1 (ABI1), a negative regulator of ABA signalling. Together, these findings reveal an important regulatory mechanism for modulating ABI1 levels by the CRK4-PUB13 module to fine-tune drought tolerance in Arabidopsis.

Osmotic stress-induced localisation switch of CBR1 from mitochondria to the endoplasmic reticulum triggers ATP production via ß-oxidation to respond to osmotic shock.

Drought and high salinity are major environmental factors that reduce plant growth and development, leading to loss of plant productivity in agriculture. Under these stress conditions, photosynthesis is greatly suppressed despite the high cellular energy cost of stress response processes. Currently, the process that allows plants to secure the energy required for osmotic stress responses remains elusive. Here, we provide evidence that cytochrome b5 reductase 1 (CBR1), a cytochrome b5 reductase, plays an important role in ATP production in response to NaCl and dehydration stresses. Overexpression and loss of function of CBR1 led to enhanced resistance and sensitivity, respectively, to osmotic stress. Upon exposure to osmotic stress, CBR1 was localised to the endoplasmic reticulum (ER) instead of to mitochondria, where it was localised under normal conditions. Transgenic plants overexpressing ER-targeted CBR1 showed enhanced resistance to osmotic stress. Moreover, CBR1-ER and CBR1-OX plants, had higher levels of ATP and unsaturated fatty acids under osmotic stress. However, these effects were abrogated by thioridazine and 2-deoxy glucose, inhibitors of ß-oxidation and glycolysis, respectively. Based on these results, we propose that ER-localised CBR1 triggers ATP production via the production and ß-oxidation of polyunsaturated fatty acids under osmotic stress.

Comparative transcriptomics reveals the role of altered energy metabolism in the establishment of single-cell C4 photosynthesis in Bienertia sinuspersici.

Single-cell C4 photosynthesis (SCC4) in terrestrial plants without Kranz anatomy involves three steps: initial CO2 fixation in the cytosol, CO2 release in mitochondria, and a second CO2 fixation in central chloroplasts. Here, we investigated how the large number of mechanisms underlying these processes, which occur in three different compartments, are orchestrated in a coordinated manner to establish the C4 pathway in Bienertia sinuspersici, a SCC4 plant. Leaves were subjected to transcriptome analysis at three different developmental stages. Functional enrichment analysis revealed that SCC4 cycle genes are coexpressed with genes regulating cyclic electron flow and amino/organic acid metabolism, two key processes required for the production of energy molecules in C3 plants. Comparative gene expression profiling of B. sinuspersici and three other species (Suaeda aralocaspica, Amaranthus hypochondriacus, and Arabidopsis thaliana) showed that the direction of metabolic flux was determined via an alteration in energy supply in peripheral chloroplasts and mitochondria via regulation of gene expression in the direction of the C4 cycle. Based on these results, we propose that the redox homeostasis of energy molecules via energy metabolism regulation is key to the establishment of the SCC4 pathway in B. sinuspersici.

Randomized phase III trial of a neoadjuvant regimen of four cycles of adriamycin plus cyclophosphamide followed by four cycles of docetaxel (AC4-D4) versus a shorter treatment of three cycles of FEC followed by three cycles of docetaxel (FEC3-D3) in node-positive breast cancer (Neo-shorter; NCT02001506).

PURPOSE: To determine whether six cycles of FEC3-D3 has a comparable efficacy to eight of AC4-D4. METHODS: The enrolled patients (pts) were clinically diagnosed with stage II or III breast cancer. The primary endpoint was a pathologic complete response (pCR), and the secondary endpoints were 3 year disease-free survival (3Y DFS), toxicities, and health-related quality of life (HRQoL). We calculated that 252 pts were needed in each treatment group to enable the detection of non-inferiority (non-inferiority margin of 10%). RESULTS: In terms of ITT analysis, 248 pts were finally enrolled. The 218 pts who completed the surgery were included in the current analysis. The baseline characteristics of these subjects were well balanced between the two arms. By ITT analysis, pCR was achieved in 15/121 (12.4%) pts in the FEC3-D3 arm and 18/126 (14.3%) in the AC4-D4 arm. With a median follow up of 64.1 months, the 3Y DFS was comparable between the two arms (75.8% in FEC3-D3 vs. 75.6% in AC4-D4). The most common adverse event (AE) was Grade 3/4 neutropenia, which arose in 27/126 (21.4%) AC4-D4 arm pts vs 23/121 (19.0%) FEC3-D3 arm cases. The primary HRQoL domains were similar between the two groups (FACT-B scores at baseline, P = 0.35; at the midpoint of NACT, P = 0.20; at the completion of NACT, P = 0.44). CONCLUSION: Six cycles of FEC3-D3 could be an alternative to eight of AC4-D4. Trial registration ClinicalTrials.gov NCT02001506. Registered December 5,2013. https://clinicaltrials.gov/ct2/show/NCT02001506.

Assembly of Human Papillomavirus 16 L1 Protein in Nicotiana benthamiana Chloroplasts into Highly Immunogenic Virus-Like Particles.

Infection with human papillomavirus (HPV) can cause cervical cancers in women, and vaccination against the virus is one of most effective ways to prevent these cancers. Two vaccines made of virus-like particles (VLPs) of HPV L1 proteins are currently commercially available. However, these HPV vaccines are highly expensive, and thus not affordable for women living in developing countries. Therefore, great demand exists to produce a cost-effective vaccine. Here, we investigate the production of self-assembled HPV16 VLPs in plants. We generated a chimeric protein composed of N-terminal 79 amino acid residues of RbcS as a long-transit peptide to target chloroplasts, the SUMO domain, and HPV16 L1 proteins. The chimeric gene was expressed in plants with chloroplast-targeted bdSENP1, a protein that specifically recognizes the SUMO domain and cleaves its cleavage site. This co-expression of bdSENP1 led to the release of HPV16 L1 from the chimeric proteins without any extra amino acid residues. HPV16 L1 purified by heparin chromatography formed VLPs that mimicked native virions. Moreover, the plant-produced HPV16 L1 VLPs elicited strong immune responses in mice without adjuvants. Thus, we demonstrated the cost-effective production of HPV16 VLPs in plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12374-023-09393-6.

Metabolic flexibility during a trophic transition reveals the phenotypic plasticity of greater duckweed (Spirodela polyrhiza 7498).

The greater duckweed (Spirodela polyrhiza 7498) exhibits trophic diversity (photoautotrophic, heterotrophic, photoheterotrophic, and mixotrophic growth) depending on the availability of exogenous organic carbon sources and light. Here, we show that the ability to transition between various trophic growth conditions is an advantageous trait, providing great phenotypic plasticity and metabolic flexibility in S. polyrhiza 7498. By comparing S. polyrhiza 7498 growth characteristics, metabolic acclimation, and cellular ultrastructure across these trophic modes, we show that mixotrophy decreases photosynthetic performance and relieves the CO2 limitation of photosynthesis by enhancing the CO2 supply through the active respiration pathway. Proteomic and metabolomic analyses corroborated that S. polyrhiza 7498 increases its intracellular CO2 and decreases reactive oxygen species under mixotrophic and heterotrophic conditions, which substantially suppressed the wasteful photorespiration and oxidative-damage pathways. As a consequence, mixotrophy resulted in a higher biomass yield than the sum of photoautotrophy and heterotrophy. Our work provides a basis for using trophic transitions in S. polyrhiza 7498 for the enhanced accumulation of value-added products.

Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana).

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM'M-UD, coupled with an artificial terminator, 3PRt. FM'M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis-acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM'M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4-6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM'M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM'M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria.

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal-anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins.

Subcellular organelles in eukaryotes are surrounded by lipid membranes. In an endomembrane system, vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles. However, organellar proteins for chloroplasts, mitochondria, the nucleus, and peroxisomes that are translated in the cytosol are directly imported into their target organelles. Chloroplasts are a plant-specific organelle with outer and inner envelope membranes, a dual-membrane structure that is similar to mitochondria. Interior chloroplast proteins translated by cytosolic ribosomes are thus translocated through TOC and TIC complexes (translocons in the outer and inner envelope of chloroplasts, respectively), with stromal ATPase motor proteins playing a critical role in pulling pre-proteins through these import channels. Over the last three decades, the identity and function of TOC/TIC components and stromal motor proteins have been actively investigated, which has shed light on the action mechanisms at a molecular level. However, there remains some disagreement over the exact composition of TIC complexes and genuine stromal motor proteins. In this review, we discuss recent findings on the mechanisms by which proteins are translocated through TOC/TIC complexes and discuss future prospects for this field of research.

Recombinant proteins of spike protein of SARS-CoV-2 with the Omicron receptor-binding domain induce production of highly Omicron-specific neutralizing antibodies.

Various vaccines have been developed to fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 pandemic. However, new variants of SARS-CoV-2 undermine the effort to fight SARS-CoV-2. Here, we produced S proteins harboring the receptor-binding domain (RBD) of the Omicron variant in plants. Plant-produced S proteins together with adjuvant CIA09A triggered strong immune responses in mice. Antibodies in serum inhibited interaction of recombinant human angiotensin-converting enzyme 2 with RBD of the Omicron variant, but not RBD of other variants. These results suggest that antibodies induced by RBD of the Omicron variant are highly specific for the Omicron RBD, but not for that of other variants.

Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples.

Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E. coli, it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1-3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E. coli. To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.

Tolerant mechanism of model legume plant Medicago truncatula to drought, salt, and cold stresses.

Legume plants produce one-third of the total yield of primary crops and are important food sources for both humans and animals worldwide. Frequent exposure to abiotic stresses, such as drought, salt, and cold, greatly limits the production of legume crops. Several morphological, physiological, and molecular studies have been conducted to characterize the response and adaptation mechanism to abiotic stresses. The tolerant mechanisms of the model legume plant Medicago truncatula to abiotic stresses have been extensively studied. Although many potential genes and integrated networks underlying the M. truncatula in responding to abiotic stresses have been identified and described, a comprehensive summary of the tolerant mechanism is lacking. In this review, we provide a comprehensive summary of the adaptive mechanism by which M. truncatula responds to drought, salt, and cold stress. We also discuss future research that need to be explored to improve the abiotic tolerance of legume plants.

SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

PtomtAPX is an autonomous lignification peroxidase during the earliest stage of secondary wall formation in Populus tomentosa Carr.

At present, a cooperative process hypothesis is used to explain the supply of enzyme (class III peroxidases and/or laccases) and substrates during lignin polymerization. However, it remains elusive how xylem cells meet the needs of early lignin rapid polymerization during secondary cell wall formation. Here we provide evidence that a mitochondrial ascorbate peroxidase (PtomtAPX) is responsible for autonomous lignification during the earliest stage of secondary cell wall formation in Populus tomentosa. PtomtAPX was relocated to cell walls undergoing programmed cell death and catalysed lignin polymerization in vitro. Aberrant phenotypes were caused by altered PtomtAPX expression levels in P. tomentosa. These results reveal that PtomtAPX is crucial for catalysing lignin polymerization during the early stages of secondary cell wall formation and xylem development, and describe how xylem cells provide autonomous enzymes needed for lignin polymerization during rapid formation of the secondary cell wall by coupling with the programmed cell death process.

Clinical Outcomes and Effectiveness of Heart Transplantation in Patients With Systemic Light-chain Cardiac Amyloidosis.

BACKGROUND: In systemic light-chain (AL) amyloidosis, cardiac involvement is a major determinant of survival; however, cardiac response is limited even after systemic treatment in a majority of patients, and some require heart transplantation. Additionally, limited information is available on specific indications for heart transplantation. We aimed to explore clinical outcomes of cardiac amyloidosis and its association with heart transplantation, including identifying factors favoring heart transplantation amenability. METHODS: We retrospectively analyzed data from patients diagnosed with AL amyloidosis with cardiac involvement between January 2007 and December 2020 at a tertiary referral center. RESULTS: Among 73 patients, 72 (99%) received systemic treatment, and 12 (16%) underwent heart transplantation. Characteristics at diagnosis were similar between heart transplant recipients and nonrecipients, although left ventricular ejection fraction tended to be lower in recipients (median 48% versus 57%, P = 0.085). Eight weeks after systemic treatment, 67% and 12% of patients achieved hematologic and brain natriuretic peptide responses. Overall survival was longer among heart transplantation recipients than nonrecipients, with 5-y survival rates of 61.1% (95% confidence interval, 25.5%-83.8%) versus 32.0% (95% confidence interval, 20.3%-44.4%; P = 0.022), respectively. Among the 34 with identifiable causes of death out of 51 deaths, 21 nonrecipients (62%) died of cardiac problems compared with none in the heart transplant recipients. Additionally, survival outcomes favored heart transplant recipients in most subgroups, including patients with higher Mayo 2004 European stage at diagnosis and with extracardiac involvement of amyloidosis. CONCLUSIONS: Heart transplantation can achieve long-term survival in appropriately selected patients with AL cardiac amyloidosis.

Production of Recombinant Active Human TGFß1 in Nicotiana benthamiana.

The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFß1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFß1, various recombinant genes containing the mature form of TGFß1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFß1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFß1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFß1. The mature TGFß1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFß1 activated TGFß1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFß1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1.

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15th and 21st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31st to 40th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41st to 50th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.

GREEN FLUORESCENT PROTEIN variants with enhanced folding are more efficiently imported into chloroplasts.

Chloroplasts and mitochondria are subcellular organelles that evolved from cyanobacteria and α-proteobacteria, respectively. Although they have their own genomes, the majority of their proteins are encoded by nuclear genes, translated by cytosolic ribosomes, and imported via outer and inner membrane translocon complexes. The unfolding of mature regions of proteins is thought to be a prerequisite for the import of the proteins into these organelles. However, it is not fully understood how protein folding properties affect their import into these organelles. In this study, we examined the import behavior of chloroplast and mitochondrial reporters with normal green fluorescent protein (GFP) and two GFP variants with enhanced folding propensity, superfolder GFP (sfGFP) and extra-superfolder GFP (esGFP), which is folded better than sfGFP. sfGFP and esGFP were less dependent on the sequence motifs of the transit peptide (TP) and import machinery during protein import into Arabidopsis (Arabidopsis thaliana) chloroplasts, compared with normal GFP. sfGFP and esGFP were efficiently imported into chloroplasts by a mutant TP with an alanine substitution in the N-terminal MLM motif, whereas the same mutant TP showed a defect in importing normal GFP into chloroplasts. Moreover, sfGFP and esGFP were efficiently imported into plastid protein import 2 (ppi2) and heat shock protein 93-V (hsp93-V) plants, which have mutations in atToc159 and Hsp93-V, respectively. In contrast, the presequence-mediated mitochondrial import of sfGFP and esGFP was severely impaired. Based on these results, we propose that the chloroplast import machinery is more tolerant to different folding states of preproteins, whereas the mitochondrial machinery is more specialized in the translocation of unfolded preproteins.