Subunit E isoform 1 of vacuolar H+-ATPase OsVHA enables post-Golgi trafficking of rice seed storage proteins.

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.

white panicle2 encoding thioredoxin z, regulates plastid RNA editing by interacting with multiple organellar RNA editing factors in rice.

Thioredoxins (TRXs) occur in plant chloroplasts as complex disulphide oxidoreductases. Although many biological processes are regulated by thioredoxins, the regulatory mechanism of chloroplast TRXs are largely unknown. Here we report a rice white panicle2 mutant caused by a mutation in the thioredoxin z gene, an orthologue of AtTRX z in Arabidopsis. white panicle2 (wp2) seedlings exhibited a high-temperature-sensitive albinic phenotype. We found that plastid multiple organellar RNA editing factors (MORFs) were the regulatory targets of thioredoxin z. We showed that OsTRX z protein physically interacts with OsMORFs in a redox-dependent manner and that the redox state of a conserved cysteine in the MORF box is essential for MORF-MORF interactions. wp2 and OsTRX z knockout lines show reduced editing efficiencies in many plastidial-encoded genes especially under high-temperature conditions. An Arabidopsis trx z mutant also exhibited significantly reduced chloroplast RNA editing. Our combined results suggest that thioredoxin z regulates chloroplast RNA editing in plants by controlling the redox state of MORFs.

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice.

A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDP-glucose 4-epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo- and hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.

WSL6 encoding an Era-type GTP-binding protein is essential for chloroplast development in rice.

KEY MESSAGE: Rice WSL6 is involved in chloroplast ribosome biogenesis and is essential for early chloroplast development. Construction of the genetic translation system is a prerequisite for chloroplast development in plants. However, the molecular mechanism underlying this process is largely unknown. Here, we isolated a white stripe leaf6 (wsl6) mutant in rice. The mutant seedlings displayed white-striped leaves that were more severe under low-temperature conditions. Transmission electron microscopy analysis showed that the wsl6 mutant was defective in early chloroplast development. Map-based cloning revealed that WSL6 encodes an Era-type guanosine-5'-triphosphate (GTP)-binding protein located in chloroplasts. Immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated an absence of 70S ribosomes in wsl6 chloroplasts. Further research showed that WSL6 binds to the 16S ribosomal RNA (rRNA) subunit of chloroplast ribosome 30S. In summary, these results show that WSL6 is essential for chloroplast ribosome biogenesis during early chloroplast development in rice.

OsNHX5-mediated pH homeostasis is required for post-Golgi trafficking of seed storage proteins in rice endosperm cells.

BACKGROUND: As the major storage protein in rice seeds, glutelins are synthesized at the endoplasmic reticulum (ER) as proglutelins and transported to protein storage vacuoles (PSVs) called PBIIs (Protein body IIs), where they are cleaved into mature forms by the vacuolar processing enzymes. However, the molecular mechanisms underlying glutelin trafficking are largely unknown. RESULTS: In this study, we report a rice mutant, named glutelin precursor accumulation6 (gpa6), which abnormally accumulates massive proglutelins. Cytological analyses revealed that in gpa6 endosperm cells, proglutelins were mis-sorted, leading to the presence of dense vesicles (DVs) and the formation paramural bodies (PMBs) at the apoplast, consequently, smaller PBII were observed. Mutated gene in gpa6 was found to encode a Na+/H+ antiporter, OsNHX5. OsNHX5 is expressed in all tissues analyzed, and its expression level is much higher than its closest paralog OsNHX6. The OsNHX5 protein colocalizes to the Golgi, the trans-Golgi network (TGN) and the pre-vacuolar compartment (PVC) in tobacco leaf epidermal cells. In vivo pH measurements indicated that the lumens of Golgi, TGN and PVC became more acidic in gpa6. CONCLUSIONS: Our results demonstrated an important role of OsNHX5 in regulating endomembrane luminal pH, which is essential for seed storage protein trafficking in rice.

FLOURY ENDOSPERM16 encoding a NAD-dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice.

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map-based cloning and complementation tests demonstrated that FLO16 encodes a NAD-dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis-related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

FLOURY SHRUNKEN ENDOSPERM1 Connects Phospholipid Metabolism and Amyloplast Development in Rice.

Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during endosperm development.

The nuclear-localized PPR protein OsNPPR1 is important for mitochondrial function and endosperm development in rice.

Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants. Recent studies revealed the functions of PPR proteins in organellar RNA metabolism and plant development, but the functions of most PPR proteins, especially PPRs localized in the nucleus, remain largely unknown. Here, we report the isolation and characterization of a rice mutant named floury and growth retardation1 (fgr1). fgr1 showed floury endosperm with loosely arranged starch grains, decreased starch and amylose contents, and retarded seedling growth. Map-based cloning showed that the mutant phenotype was caused by a single nucleotide substitution in the coding region of Os08g0290000. This gene encodes a nuclear-localized PPR protein, which we named OsNPPR1, that affected mitochondrial function. In vitro SELEX and RNA-EMSAs showed that OsNPPR1 was an RNA protein that bound to the CUCAC motif. Moreover, a number of retained intron (RI) events were detected in fgr1. Thus, OsNPPR1 was involved in regulation of mitochondrial development and/or functions that are important for endosperm development. Our results provide novel insights into coordinated interaction between nuclear-localized PPR proteins and mitochondrial function.

GOLGI TRANSPORT 1B Regulates Protein Export from the Endoplasmic Reticulum in Rice Endosperm Cells.

Coat protein complex II (COPII) mediates the first step of anterograde transport of newly synthesized proteins from the endoplasmic reticulum (ER) to other endomembrane compartments in eukaryotes. A group of evolutionarily conserved proteins (Sar1, Sec23, Sec24, Sec13, and Sec31) constitutes the basic COPII coat machinery; however, the details of how the COPII coat assembly is regulated remain unclear. Here, we report a protein transport mutant of rice (Oryza sativa), named glutelin precursor accumulation4 (gpa4), which accumulates 57-kD glutelin precursors and forms two types of ER-derived abnormal structures. GPA4 encodes the evolutionarily conserved membrane protein GOT1B (also known as GLUP2), homologous to the Saccharomyces cerevisiae GOT1p. The rice GOT1B protein colocalizes with Arabidopsis thaliana Sar1b at Golgi-associated ER exit sites (ERESs) when they are coexpressed in Nicotiana benthamiana Moreover, GOT1B physically interacts with rice Sec23, and both proteins are present in the same complex(es) with rice Sar1b. The distribution of rice Sar1 in the endomembrane system, its association with rice Sec23c, and the ERES organization pattern are significantly altered in the gpa4 mutant. Taken together, our results suggest that GOT1B plays an important role in mediating COPII vesicle formation at ERESs, thus facilitating anterograde transport of secretory proteins in plant cells.

OsNDUFA9 encoding a mitochondrial complex I subunit is essential for embryo development and starch synthesis in rice.

KEY MESSAGE: Loss of function of a mitochondrial complex I subunit (OsNDUFA9) causes abnormal embryo development and affects starch synthesis by altering the expression of starch synthesis-related genes and proteins. Proton-pumping NADH: ubiquinone oxidoreductase (also called complex I) is thought to be the largest and most complicated enzyme of the mitochondrial respiratory chain. Mutations of complex I subunits have been revealed to link with a number of growth inhibitions in plants. However, the function of complex I subunits in rice remains unclear. Here, we isolated a rice floury endosperm mutant (named flo13) that was embryonic lethal and failed to germinate. Semi-thin sectioning analysis showed that compound starch grain development in the mutant was greatly impaired, leading to significantly compromised starch biosynthesis and decreased 1000-grain weight relative to the wild type. Map-based cloning revealed that FLO13 encodes an accessory subunit of complex I protein (designated as OsNDUFA9). A single nucleotide substitution (G18A) occurred in the first exon of OsNDUFA9, introducing a premature stop codon in the flo13 mutant gene. OsNDUFA9 was ubiquitously expressed in various tissues and the OsNDUFA9 protein was localized to the mitochondria. Quantitative RT-PCR and protein blotting indicated loss of function of OsNDUFA9 altered gene expression and protein accumulation associated with respiratory electron chain complex in the mitochondria. Moreover, transmission electron microscopic analysis showed that the mutant lacked obvious mitochondrial cristae structure in the mitochondria of endosperm cell. Our results demonstrate that the OsNDUFA9 subunit of complex I is essential for embryo development and starch synthesis in rice endosperm.

OsPKpα1 encodes a plastidic pyruvate kinase that affects starch biosynthesis in the rice endosperm.

Pyruvate kinase (PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice (Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK (PKp). OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein-protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts.

The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice.

Mutations of ribosomal proteins (RPs) are known to cause developmental abnormalities in yeast, mammals, and dicotyledonous plants; however, their effects have not been studied in rice. Here, we identifiy a ribosomal biogenesis mutant, rice minute-like1 (rml1) that displays a minute phenotype as evidenced by retarded growth and defects in the vascular system. We determine that RML1 encodes a ribosome large subunit protein 3B (RPL3B) in rice by means of map-based cloning and genetic complementation. RPL3B is abundantly expressed in all the tissues, whereas RPL3A, another RPL3 gene family member, is expressed at low levels. Notably, the expression level of RPL3A in the rml1 mutant is similar to that in the wild-type, suggesting that RPL3A provides no functional compensation for RPL3B in rml1 plants. Ribosomal profiles show that mutation of RPL3B leads to a significant reduction in free 60S ribosomal subunits and polysomes, indicating a ribosomal insufficiency in the rml1 mutant. Our results demonstrate that the ribosomal protein gene RPL3B is required for maintaining normal leaf morphology and plant architecture in rice through its regulation of ribosome biogenesis.

Corrigendum: Integrated GWAS and transcriptomic analysis reveal the candidate salt-responding genes regulating Na+/K+ balance in barley (Hordeum vulgare L.).

[This corrects the article DOI: 10.3389/fpls.2022.1004477.].

Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants.

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants. Previous studies have established that tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) play essential roles in plastid retrograde signaling during early chloroplast biogenesis; however, their functional relationship remains unknown. In this study, we generated a series of rice TPB-related gun (genome uncoupled) mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon (NF; a noncompetitive inhibitor of carotenoid biosynthesis) and/or lincomycin (Lin; a specific inhibitor of plastid translation). We show that under NF treatment, expression of plastid-encoded polymerase (PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants. We further demonstrate that the derepressed expression of PEP-transcribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome. In addition, we show that expression of photosynthesis-associated nuclear genes (PhANGs) and PEP-transcribed genes is correlated in the rice TPB-related gun mutants, with or without NF or Lin treatment. A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants. Moreover, we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants. Further, we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling. Taken together, our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

Integrated GWAS and transcriptomic analysis reveal the candidate salt-responding genes regulating Na+/K+ balance in barley (Hordeum vulgare L.).

Salt stress is one of the main abiotic stresses affecting crop yield and quality. Barley has strong salt tolerance, however, the underlying genetic basis is not fully clear, especially in the seedling stage. This study examined the ionic changes in barley core germplasms under the control and salt conditions. Genome-wide association study (GWAS) analysis revealed 54 significant SNPs from a pool of 25,342 SNPs distributed in 7 chromosomes (Chr) of the Illumina Barley 50K SNP array. These SNPs are associated with ion homeostasis traits, sodium (Na+) and potassium (K+) content, and Na+/K+ ratio representing five genomic regions on Chr 2, 4, 5, 6, and 7 in the leaves of worldwide barley accessions. And there are 3 SNP peaks located on the Chr 4, 6, and 7, which could be the "hot spots" regions for mining and identifying candidate genes for salt tolerance. Furthermore, 616 unique candidate genes were screened surrounding the significant SNPs, which are associated with transport proteins, protein kinases, binding proteins, and other proteins of unknown function. Meanwhile, transcriptomic analysis (RNA-Seq) was carried out to compare the salt-tolerant (CM72) and salt-sensitive (Gairdner) genotypes subjected to salt stress. And there was a greater accumulation of differentially expressed genes(DEGs) in Gairdner compared to CM72, mainly enriched in metabolic pathway, biosynthesis of secondary metabolites, photosynthesis, signal transduction,emphasizing the different transcriptional response in both genotypes following salt exposure. Combined GWAS and RNA-Seq analysis revealed 5 promising salt-responding genes (PGK2, BASS3, SINAT2, AQP, and SYT3) from the hot spot regions, which were verified between the salt-tolerant and salt-sensitive varieties by qRT-PCR. In all, these results provide candidate SNPs and genes responsible for salinity responding in barley, and a new idea for studying such genetic basis in similar crops.

ENLARGED STARCH GRAIN1 affects amyloplast development and starch biosynthesis in rice endosperm.

Cereal crops accumulate large amounts of starch which is synthesized and stored in amyloplasts in the form of starch grains (SGs). Despite significant progress in deciphering starch biosynthesis, our understanding of amyloplast development in rice (Oryza sativa) endosperm remains largely unknown. Here, we report a novel rice floury mutant named enlarged starch grain1 (esg1). The mutant has decreased starch content, altered starch physicochemical properties, slower grain-filling rate and reduced 1000-grain weight. A distinctive feature in esg1 endosperm is that SGs are much larger, mainly due to an increased number of starch granules per SG. Spherical and loosely assembled granules, together with those weakly stained SGs may account for decreased starch content in esg1. Map-based cloning revealed that ESG1 encodes a putative permease subunit of a bacterial-type ABC (ATP-binding cassette) lipid transporter. ESG1 is constitutively expressed in various tissues. It encodes a protein localized to the chloroplast and amyloplast membranes. Mutation of ESG1 causes defective galactolipid synthesis. The overall study indicates that ESG1 is a newly identified protein affecting SG development and subsequent starch biosynthesis, which provides novel insights into amyloplast development in rice.

FLOURY ENDOSPERM11 encoding a plastid heat shock protein 70 is essential for amyloplast development in rice.

Mutations of stromal Hsp70 cause chloroplast developmental abnormalities and knockout mutants of stromal Hsp70 usually exhibit protein import deficiencies. However, their effects have not been studied in amyloplast development. Here, we identified an amyloplast abnormal development mutant, floury endosperm11 (flo11) that exhibited an opaque phenotype in the inner core and the periphery of grains. Semi-thin section revealed defective amyloplast development in the flo11 endosperm. Map-based cloning and subsequent complementation test demonstrated that FLO11 encoded a plastid-localized heat shock protein 70 (OsHsp70cp-2). OsHsp70cp-2 was abundantly expressed in developing endosperm, whereas its paralogous gene OsHsp70cp-1 was mainly expressed in photosynthetic tissues. Ectopic expression of OsHsp70cp-1 under the control of OsHsp70cp-2 promoter rescued the mutant phenotype of flo11. Moreover, simultaneous knockdown of both OsHsp70cp genes resulted in white stripe leaves and opaque endosperm. BiFC and Co-IP assays revealed that OsHsp70cp-2 was associated with Tic complex. Taken together, OsHsp70cp-2 may regulate protein import into amyloplasts, which is essential for amyloplast development in rice.

SGD1, a key enzyme in tocopherol biosynthesis, is essential for plant development and cold tolerance in rice.

Tocopherols, a group of Vitamin E compounds, are essential components of the human diet. In contrast to well documented roles in animals, the functions of tocopherols in plants are less understood. In this study, we characterized two allelic rice dwarf mutant lines designated sgd1-1 and sgd1-2 (small grain and dwarf1). Histological observations showed that the dwarf phenotypes were mainly due to cell elongation defects. A map-based cloning strategy and subsequent complementation test showed that SGD1 encodes homogentisate phytyltransferase (HPT), a key enzyme in tocopherol biosynthesis. Mutation of SGD1 resulted in tocopherol deficiency in both sgd1mutants. No oxidant damage was detected in the sgd1 mutants. Further analysis showed that sgd1-2 was hypersensitive to cold stress. Our results indicate that SGD1 is essential for plant development and cold tolerance in rice.