Consecutive multimaterial printing of biomimetic ionic hydrogel power sources with high flexibility and stretchability.

Electric eel is an excellent example to harness ion-concentration gradients for sustainable power generation. However, current strategies to create electric-eel-inspired power sources commonly involve manual stacking of multiple salinity-gradient power source units, resulting in low efficiency, unstable contact, and poor flexibility. Here we propose a consecutive multimaterial printing strategy to efficiently fabricate biomimetic ionic hydrogel power sources with a maximum stretchability of 137%. The consecutively-printed ionic hydrogel power source filaments showed seamless bonding interface and can maintain stable voltage outputs for 1000 stretching cycles at 100% strain. With arrayed multi-channel printhead, power sources with a maximum voltage of 208 V can be automatically printed and assembled in parallel within 30 min. The as-printed flexible power source filaments can be woven into a wristband to power a digital wristwatch. The presented strategy provides a tool to efficiently produce electric-eel-inspired ionic hydrogel power sources with great stretchability for various flexible power source applications.

Ecotoxicological effects of polyethylene microplastics and lead (Pb) on the biomass, activity, and community diversity of soil microbes.

Microplastics and heavy metals are ubiquitous and persistent contaminants that are widely distributed worldwide, yet little is known about the effects of their interaction on soil ecosystems. A soil incubation experiment was conducted to investigate the individual and combined effects of polyethylene microplastics (PE-MPs) and lead (Pb) on soil enzymatic activities, microbial biomass, respiration rate, and community diversity. The results indicate that the presence of PE-MPs notably reduced soil pH and elevated soil Pb bioavailability, potentially exacerbated the combined toxicity on the biogeochemical cycles of soil nutrients, microbial biomass carbon and nitrogen, and the activities of soil urease, sucrase, and alkaline phosphatase. Soil CO2 emissions increased by 7.9% with PE-MPs alone, decreased by 46.3% with single Pb, and reduced by 69.4% with PE-MPs and Pb co-exposure, compared to uncontaminated soils. Specifically, the presence of PE-MPs and Pb, individually and in combination, facilitated the soil metabolic quotient, leading to reduced microbial metabolic efficiency. Moreover, the addition of Pb and PE-MPs modified the composition of the microbial community, leading to the enrichment of specific taxa. Tax4Fun analysis showed the effects of Pb, PE-MPs and their combination on the biogeochemical processes and ecological functions of microbes were mainly by altering amino acid metabolism, carbohydrate metabolism, membrane transport, and signal transduction. These findings offer valuable insights into the ecotoxicological effects of combined PE-MPs and Pb on soil microbial dynamics, reveals key assembly mechanisms and environmental drivers, and highlights the potential threat of MPs and heavy metals to the multifunctionality of soil ecosystems.

Bioactive 3D Electrohydrodynamic Printed Lattice Architectures Augment Tenogenesis of Tendon Stem/Progenitor Cells.

Tendon defect repair remains a tough clinical procedure that hinders functional motion in patients. Electrohydrodynamic (EHD) three-dimensional (3D) printing, as a novel strategy, can controllably fabricate biomimetic micro/nanoscale architecture, but the hydrophobic and bioinert nature of polymers might be adverse to cell-material interplay. In this work, 3D EHD printed polycaprolactone (PCL) was immobilized on basic fibroblast growth factor (bFGF) using polydopamine (PDA), and the proliferation and tenogenic differentiation of tendon stem/progenitor cells (TSPCs) in vitro was researched. A subcutaneous model was established to evaluate the effects of tenogenesis and immunomodulation. We then investigated the in situ implantation and immunomodulation effects in an Achilles tendon defect model. After immobilization of bFGF, the scaffolds profoundly facilitated proliferation and tenogenic differentiation; however, PDA had only a proliferative effect. Intriguingly, the bFGF immobilized on EHD printed PCL indicated a synergistic effect on the highest expression of tenogenic gene and protein markers at 14 days, and the tenogenesis may be induced by activating the transforming growth factor-ß (TGF-ß) signal pathway in vitro. The subcutaneous engraftment study confirmed a tendon-like structure, similar to that of the native tendon, as well as an M2 macrophage polarization effect. Additionally, the bioactive scaffold exhibited superior efficacy in new collagen formation and repair of Achilles tendon defects. Our study revealed that the topographic cues alone were insufficient to trigger tenogenic differentiation, requiring appropriate chemical signals, and that appropriate immunomodulation was conducive to tenogenesis. The tenogenesis of TSPCs on the bioactive scaffold may be correlated with the TGF-ß signal pathway and M2 macrophage polarization.

Calcium phosphate coating enhances osteointegration of melt electrowritten scaffold by regulating macrophage polarization.

The osteoimmune microenvironment induced by implants plays a significant role in bone regeneration. It is essential to efficiently and timely switch the macrophage phenotype from M1 to M2 for optimal bone healing. This study examined the impact of a calcium phosphate (CaP) coating on the physiochemical properties of highly ordered polycaprolactone (PCL) scaffolds fabricated using melt electrowritten (MEW). Additionally, it investigated the influence of these scaffolds on macrophage polarization and their immunomodulation on osteogenesis. The results revealed that the CaP coated PCL scaffold exhibited a rougher surface topography and higher hydrophilicity in comparison to the PCL scaffold without coating. Besides, the surface morphology of the coating and the release of Ca2+ from the CaP coating were crucial in regulating the transition of macrophages from M1 to M2 phenotypes. They might activate the PI3K/AKT and cAMP-PKA pathways, respectively, to facilitate M2 polarization. In addition, the osteoimmune microenvironment induced by CaP coated PCL could not only enhance the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro but also promote the bone regeneration in vivo. Taken together, the CaP coating can be employed to control the phenotypic switching of macrophages, thereby creating a beneficial immunomodulatory microenvironment that promotes bone regeneration.

Coaxial Electrohydrodynamic Printing of Microscale Core-Shell Conductive Features for Integrated Fabrication of Flexible Transparent Electronics.

Reliable insulation of microscale conductive features is required to fabricate functional multilayer circuits or flexible electronics for providing specific physical/chemical/electrical protection. However, the existing strategies commonly rely on manual assembling processes or multiple microfabrication processes, which is time-consuming and a great challenge for the fabrication of flexible transparent electronics with microscale features and ultrathin thickness. Here, we present a novel coaxial electrohydrodynamic (CEHD) printing strategy for the one-step fabrication of microscale flexible electronics with conductive materials at the core and insulating material at the outer layer. A finite element analysis (FEA) method is established to simulate the CEHD printing process. The extrusion sequence of the conductive and insulating materials during the CEHD printing process shows little effect on the morphology of the core-shell filaments, which can be achieved on different flexible substrates with a minimum conductive line width of 32 ± 3.2 µm, a total thickness of 53.6 ± 4.8 µm, and a conductivity of 0.23 × 107 S/m. The thin insulating layer can provide the inner conductive filament enough protection in 3D, which endows the resultant microscale core-shell electronics with good electrical stability when working in different chemical solvent solutions or under large deformation conditions. Moreover, the presented CEHD printing strategy offers a unique capability to sequentially fabricate an insulating layer, core-shell conductive pattern, and exposed electrodes by simply controlling the material extrusion sequence. The resultant large-area transparent electronics with two-layer core-shell patterns exhibit a high transmittance of 98% and excellent electrothermal performance. The CEHD-printed flexible microelectrode array is successfully used to record the electrical signals of beating mouse hearts. It can also be used to fabricate large-area flexible capacitive sensors to accurately measure the periodical pressure force. We envision that the present CEHD printing strategy can provide a promising tool to fabricate complex three-dimensional electronics with microscale resolution, high flexibility, and multiple functionalities.

Development of Electro-Conductive Composite Bioinks for Electrohydrodynamic Bioprinting with Microscale Resolution.

Bioprinting has attracted extensive attention in the field of tissue engineering due to its unique capability in constructing biomimetic tissue constructs in a highly controlled manner. However, it is still challenging to reproduce the physical and structural properties of native electroactive tissues due to the poor electroconductivity of current bioink systems as well as the limited printing resolution of conventional bioprinting techniques. In this work, an electro-conductive hydrogel is prepared by introducing poly (3,4-ethylene dioxythiophene): poly (styrene sulfonate) (PEDOT: PSS) into an RGD (GGGGRGDSP)-functionalized alginate and fibrin system (RAF), and then electrohydrodynamic (EHD)-bioprinted to form living tissue constructs with microscale resolution. The addition of 0.1 (w/v%) PEDOT: PSS increases the electroconductivity to 1.95 ± 0.21 S m-1 and simultaneously has little effect on cell viability. Compared with pure RAF bioink, the presence of PEDOT: PSS expands the printable parameters for EHD-bioprinting, and hydrogel filaments with the smallest feature size of 48.91 ± 3.44 µm can be obtained by further optimizing process parameters. Furthermore, the EHD-bioprinted electro-conductive living tissue constructs with improved resolution show good viability (>85%). The synergy of the advanced electro-conductive hydrogel and EHD-bioprinting presented here may provide a promising approach for engineering electro-conductive and cell-laden constructs for electroactive tissue engineering.

Electrohydrodynamic Printing of Microfibrous Architectures with Cell-Scale Spacing for Improved Cellular Migration and Neurite Outgrowth.

Electrohydrodynamic (EHD) printing provides unparalleled opportunities in fabricating microfibrous architectures to direct cellular orientation. However, it faces great challenges in depositing orderly microfibers with cell-scale spacing due to inherent fiber-fiber electrostatic interactions. Here a finite element method is established to analyze the electrostatic forces induced on the EHD-printed microfibers and the relationship between the fiber diameter and spacing for parallel deposition of EHD-printed microfibers is revealed theoretically and experimentally. It is found that uniform fiber arrangement can be achieved when the fiber spacing is five times larger than the fiber diameter. This finding enables the successful printing of parallel fibrous architectures with a fiber diameter of 4.9 ± 0.1 µm and a cell-scale fiber spacing of 25.6 ± 1.9 µm. The resultant microfibrous architectures exhibit unique capability to direct cellular alignment and enhance cellular density and migration as the fiber spacing decreases from 100 to 25 µm. The EHD-printed parallel microfibers with cell-scale spacing are found to improve the outgrowth length of neurites and accelerate the migration of Schwann cells from Dorsal Root Ganglion spheres, which facilitate the formation of densely-arranged and highly-aligned cellular constructs. The presented method is promising to produce biomimetic microfibrous architectures for functional nerve regeneration.

Bioprinted living tissue constructs with layer-specific, growth factor-loaded microspheres for improved enthesis healing of a rotator cuff.

Substantial challenges remain in constructing the native tendon-to-bone interface for rotator cuff healing owing to the enthesis tissues' highly organized structural and compositional gradients. Herein, we propose to bioprint living tissue constructs with layer-specific growth factors (GFs) to promote enthesis regeneration by guiding the zonal differentiation of the loaded stem cells in situ. The sustained release of tenogenic, chondrogenic, and osteogenic GFs was achieved via microsphere-based delivery carriers embedded in the bioprinted constructs. Compared to the basal construct without GFs, the layer-specific tissue analogs realized region-specific differentiation of stem cells in vitro. More importantly, bioprinted living tissue constructs with layer-specific GFs rapidly enhanced the enthesis regeneration in a rabbit rotator cuff tear model in terms of biomechanical restoration, collagen deposition, and alignment, showing gradient interface of fibrocartilage structures with aligned collagen fibrils and an ultimate load failure of 154.3 ± 9.5 N resembling those of native enthesis tissues in 12 weeks. This exploration provides a feasible strategy to engineer living tissue constructions with region-specific differentiation potentials for the functional repair of gradient enthesis tissues. STATEMENT OF SIGNIFICANCE: Previous studies that employed acellular layer-specific scaffolds or stem cells for the reconstruction of the rotator cuff faced challenges due to their insufficient capability to rebuild the anisotropic compositional and structural gradients of native enthesis tissues. This manuscript proposed a living tissue construct with layer-specific, GFs-loaded µS, which can direct in situ and region-specific differentiation of the embedded stem cells to tenogenic, chondrogenic, and osteogenic lineages for functional regeneration of the enthesis tissues. This bioprinted living tissue construct with the unique capability to reduce fibrovascular scar tissue formation and simultaneously facilitate enthesis tissue remodeling might provide a promising strategy to repair complex and gradient tissues in the future.

OsWHY1 Interacts with OsTRX z and is Essential for Early Chloroplast Development in Rice.

WHIRLY (WHY) family proteins, a small family of single-stranded DNA (ssDNA) binding proteins, are widely found in plants and have multiple functions to regulate plant growth and development. However, WHY in rice has received less attention. In this study, we continued our previous study on OsTRX z that is important for chloroplast development. OsTRX z was discovered to interact with OsWHY1, which was confirmed using yeast two-hybrid, pull-down, and BiFC assays. Subsequently, the oswhy1 mutants were obtained by CRISPR/Cas9, which exhibited an albino phenotype and died after the three-leaf stage. Consistent with this albino phenotype, low amounts of Chl a, Chl b, and Car were detected in the oswhy1-1 mutant. Moreover, the oswhy1-1 mutant had chloroplasts with disrupted architecture and no stacked grana and thylakoid membranes. Subcellular localization showed that the OsWHY1-GFP fusion protein was targeted to the chloroplast. What's more, OsWHY1 was found to be preferentially expressed in young leaves and was involved in chloroplast RNA editing and splicing. Mutation of OsWHY1 significantly affected the expression of chloroplast and ribosome development-related and chlorophyll synthesis-related genes. In conclusion, OsWHY1 contributes to early chloroplast development and normal seedling survival in rice. These results will further elucidate the molecular mechanism of chloroplast development and expand our understanding of WHY1 functions.

Effect of electrohydrodynamic printing scaffold with different spacing on chondrocyte dedifferentiation.

Background: Osteoarthritis (OA) is a common degenerative disease. Chondrocyte dedifferentiation can accelerate the progress of OA. Three-dimensional printing (3DP) is widely used in tissue regeneration applications. A three-dimensional (3D) culture system with 3D printed scaffolds could reduce the dedifferentiation of chondrocytes during passages, which would be a potential method for chondrocyte expansion. Methods: The viability and proliferation of chondrocytes on scaffolds and effects of scaffolds with 100, 150, 200, 250 or 300 µm spacing on chondrocyte dedifferentiation were analyzed in vitro. The morphology of scaffolds and cell/scaffold constructs was observed by scanning electron microscopy (SEM). Glycosaminoglycan (GAG) was evaluated by Alcian blue staining. The effects of different spacing on chondrocyte dedifferentiation were evaluated by the messenger RNA (mRNA) and protein levels of cartilage-related genes. Results: With more binding sites, the proliferation and viability of chondrocytes on scaffolds with 100 and 150 µm spacing were better than those with 200, 250 and 300 µm spacing on day 1, but this advantage diminished over time. The histology and quantitative real-time polymerase chain reaction (qRT-PCR) results showed that 200 µm spacing inhibits chondrocyte dedifferentiation better. Conclusions: 3D printed scaffolds with 200 µm spacing can inhibit chondrocyte dedifferentiation, providing a basis for the future study of 3D printed scaffolds as an effective method for chondrocyte expansion.

The rice LRR-like1 protein YELLOW AND PREMATURE DWARF 1 is involved in leaf senescence induced by high light.

Senescence in plants is induced by endogenous physiological changes and exogenous stresses. In this study, we isolated two alleles of a novel rice (Oryza sativa) mutant, yellow and premature dwarf 1 (ypd1). The ypd1 mutants exhibited a yellow and dwarf phenotype from germination, and premature senescence starting at tillering. Moreover, the ypd1 mutants were sensitive to high light, which accelerated cell death and senescence. Consistent with their yellow phenotype, the ypd1 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. TUNEL assays together with histochemical staining demonstrated that ypd1 mutants showed cell death and that they accumulated reactive oxygen species. The ypd1 mutants also showed increased expression of genes associated with senescence. Map-based cloning revealed a G→A substitution in exon 6 (ypd1-1) and exon 13 (ypd1-2) of LOC_Os06g13050 that affected splicing and caused premature termination of the encoded protein. YPD1 was found to be preferentially expressed in the leaf and it encodes a LRR-like1 protein. Complementation, overexpression, and targeted deletion confirmed that the mutations in YPD1 caused the ypd1 phenotype. YPD1 was localized on the chloroplast membrane. Our results thus demonstrate that the novel rice LRR-like1 protein YPD1 affects chloroplast development and leaf senescence.

Primary leaf-type ferredoxin 1 participates in photosynthetic electron transport and carbon assimilation in rice.

Ferredoxins (Fds) play a crucial role in photosynthesis by regulating the distribution of electrons to downstream enzymes. Multiple Fd genes have been annotated in the Oryza sativa L. (rice) genome; however, their specific functions are not well understood. Here, we report the functional characterization of rice Fd1. Sequence alignment, phylogenetic analysis of seven rice Fd proteins and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that rice Fd1 is a primary leaf-type Fd. Electron transfer assays involving NADP+ and cytochrome c indicated that Fd1 can donate electrons from photosystem I (PSI) to ferredoxin-NADP+ reductase. Loss-of-function fd1 mutants showed chlorosis and seedling lethality at the three-leaf stage. The deficiency of Fd1 impaired photosynthetic electron transport, which affected carbon assimilation. Exogenous glucose treatment partially restored the mutant phenotype, suggesting that Fd1 plays an important role in photosynthetic electron transport in rice. In addition, the transcript levels of Fd-dependent genes were affected in fd1 mutants, and the trend was similar to that observed in fdc2 plants. Together, these results suggest that OsFd1 is the primary Fd in photosynthetic electron transport and carbon assimilation in rice.

Short-term stress from high light and high temperature triggers transcriptomic changes in the local lesions 1 rice mutant.

High light and high temperature (HLHT) stress induces the production of damaging reactive oxygen species (ROS) in many plants. Recently, we described a HLHT-sensitive rice (Oryza sativa) mutant, local lesions (ls1), that exhibits local lesions under HLHT, due to DNA damage and excess ROS accumulation. Here, we determined that an HLHT treatment induced the local lesion phenotype in ls1 within 6 h. Corroborating this result, we found that transient HLHT treatment influenced the expression of many genes in the ls1 mutant, while affecting the growth and development of young leaves.

DNA damage and reactive oxygen species cause cell death in the rice local lesions 1 mutant under high light and high temperature.

High light and high temperature (HLHT) stress may become more frequent and severe as the climate changes, affecting crop growth and resulting in reduced production. However, the mechanism of the response to HLHT stress in rice is not yet fully understood. In the present study, we screened a rice mutant library using HLHT conditions and isolated an HLHT-sensitive mutant, local lesions 1 (ls1), which showed decreased pigment contents, defective stomata and chloroplasts, and a local lesions phenotype under HLHT. We characterized and cloned LS1 by map-based cloning and genetic complementation. LS1 encodes the A subunit of the RNase H2 complex (RNASEH2A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and comet assays indicated that mutation of LS1 led to severe DNA damage under HLHT stress. Furthermore, we found excessive reactive oxygen species (ROS) accumulation in the ls1 mutant under HLHT stress. Exogenous antioxidants eased the local lesions phenotype of the ls1 mutant under HLHT. DNA damage caused by HLHT stress induces ROS accumulation, which causes the injury and apoptosis of leaf cells in the ls1 mutant. These results enhance our understanding of the regulatory mechanism in the response to HLHT stress in higher plants.

Knocking Out the Gene RLS1 Induces Hypersensitivity to Oxidative Stress and Premature Leaf Senescence in Rice.

Improving a plant's level of tolerance to oxidative stress can frequently also enhance its tolerance to several other abiotic stresses. Here, a screen of a japonica type rice T-DNA insertion mutant library identified a highly oxidative stress-sensitive mutant. The line exhibited premature leaf senescence, starting at the three-leaf stage, and the symptoms were particularly severe from the five-leaf stage onwards. The leaves progressively lost chlorophyll, suffered protein degradation and were compromised with respect to their photosynthetic activity; their leaf mesophyll and bulliform cells became shrunken, and several senescence-associated genes (SAGs), senescence-associated transcription factor genes (SATFs) and autophagy-related genes (ATGs) were progressively up-regulated. The product of the gene inactivated by the mutation, identified via positional cloning, was putatively a ubiquitin-conjugating enzyme. The gene was denoted here as RLS1 (reactive oxygen species-sensitive leaf senescence1). The phenotype of plants in which RLS1 was knocked down using RNA interference was comparable to that of the rls1 mutant. A comparative analysis of the knock-out line and the wild type leaves showed that the former accumulated more hydrogen peroxide and more malondialdehyde, expressed a heightened level of superoxide dismutase activity and a decreased level of catalase activity, and exhibited an altered transcriptional profile with respect to several SAGs, SATFs and ATGs, and that these effects were magnified when the plants were exposed to oxidative stress. The product of RLS1 is presumed to be a critical component of the rice oxidative stress response and is involved in ROS (reactive oxygen species)-mediated leaf senescence.

The rice white green leaf 2 gene causes defects in chloroplast development and affects the plastid ribosomal protein S9.

BACKGROUND: Plastid ribosomal proteins (PRPs) play important roles in the translation of key proteins involved in chloroplast development and photosynthesis. PRPs have been widely studied in many plant species; however, few studies have investigated their roles in rice. RESULT: In the present study, we used ethyl methane sulfonate mutagenesis and obtained a novel rice mutant called white green leaf 2 (wgl2). The wgl2 mutants exhibited an albino phenotype from germination through the three-leaf stage, and then gradually transitioned to green through the later developmental stages. Consistent with this albino phenotype, wgl2 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Map-based cloning and DNA sequencing analyses of wgl2 revealed a single-nucleotide substitution (G to T) in the first exon of LOC_Os03g55930, which resulted in a substitution of glycine 92 to valine (G92 V). WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. Complementation and targeted deletion experiments confirmed that the point mutation in WGL2 is responsible for the wgl2 mutant phenotype. WGL2 is preferentially expressed in the leaf, and mutating WGL2 led to obvious changes in the expression of genes related to chlorophyll biosynthesis, photosynthesis, chloroplast development, and ribosome development compared with wild-type. CONCLUSIONS: WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. WGL2 is essential for early chloroplast development in rice. These results facilitate research that will further uncover the molecular mechanism of chloroplast development.

FRUCTOKINASE-LIKE PROTEIN 1 interacts with TRXz to regulate chloroplast development in rice.

Chloroplast genes are transcribed by the plastid-encoded RNA polymerase (PEP) or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS (FLNs) are phosphofructokinase-B (PfkB)-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLN activity in rice remain elusive. Previously, we identified and characterized a heat-stress sensitive albino (hsa1) mutant in rice. Map-based cloning revealed that HSA1 encodes a putative OsFLN2. Here, we further demonstrated that knockdown or knockout of the OsFLN1, a close homolog of HSA1/OsFLN2, considerably inhibits chloroplast biogenesis and the fln1 knockout mutants, created by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associate protein 9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN1 localizes to the chloroplast. Yeast two-hybrid, pull-down and bimolecular fluorescence complementation experiments revealed that OsFLN1 and HSA1/OsFLN2 interact with THIOREDOXINZ (OsTRXz) to regulate chloroplast development. In agreement with this, knockout of OsTRXz resulted in a similar albino and seedling lethality phenotype to that of the fln1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln1 and trxz mutants, indicating that loss of OsFLN1, HSA1/OsFLN2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN1 and HSA1/OsFLN2 contribute to chloroplast biogenesis and plant growth.

The newly identified heat-stress sensitive albino 1 gene affects chloroplast development in rice.

High temperature, a major abiotic stress, significantly affects the yield and quality of crops in many parts of the world. Components of the photosynthetic apparatus are highly susceptible to thermal damage. Although the responses to acute heat stress have been studied intensively, the mechanisms that regulate chloroplast development under heat stress remain obscure, especially in crop plants. Here, we cloned and characterized the gene responsible for the heat-sensitive albino1 (hsa1) mutation in rice (Oryza sativa). The hsa1 mutant harbors a recessive mutation in a gene encoding fructokinase-like protein2 (FLN2); the mutation causes a premature stop codon and results in a severe albino phenotype, with defects in early chloroplast development. The color of hsa1 mutant plants gradually changed from albino to green at later stages of development at various temperatures and chloroplast biogenesis was strongly delayed at high temperature (32 °C). HSA1 expression was strongly reduced in hsa1 plants compared to wild type (WT). HSA1 localizes to the chloroplast and regulates chloroplast development. An HSA1 deletion mutant induced by CRISPR/Cas9 was heat sensitive but had a faster greening phenotype than the original hsa1 allele at all temperatures. RNA and protein levels of plastid-encoded RNA polymerase-dependent plastid genes were markedly reduced in hsa1 plants compared to WT. These results demonstrated that HSA1 plays important roles in chloroplast development at early stages, and functions in protecting chloroplasts under heat stress at later stages in rice.