Simultaneous Improvement of Grain Yield and Quality through Manipulating Two Type C G Protein Gamma Subunits in Rice.

Heterotrimeric G protein signaling is an evolutionarily conserved mechanism in diverse organisms that mediates intracellular responses to external stimuli. In rice, the G proteins are involved in the regulation of multiple important agronomic traits. In this paper, we present our finding that two type C G protein gamma subunits, DEP1 and GS3, antagonistically regulated grain yield and grain quality. The DEP1 gene editing we conducted, significantly increased the grain number per panicle but had a negative impact on taste value, texture properties, and chalkiness-related traits. The GS3 gene editing decreased grain number per panicle but significantly increased grain length. In addition, the GS3 gene-edited plants showed improved taste value, appearance, texture properties, and Rapid Visco Analyser (RVA) profiles. To combine the advantages of both gs3 and dep1, we conducted a molecular design breeding at the GS3 locus of a "super rice" variety, SN265, which has a truncated dep1 allele with erect panicle architecture, high-yield performance, and which is of mediocre eating quality. The elongated grain size of the sn265/gs3 gene-edited plants further increased the grain yield. More importantly, the texture properties and RVA profiles were significantly improved, and the taste quality was enhanced. Beyond showcasing the combined function of dep1 and gs3, this paper presents a strategy for the simultaneous improvement of rice grain yield and quality through manipulating two type C G protein gamma subunits in rice.

Chlorosis seedling lethality 1 encoding a MAP3K protein is essential for chloroplast development in rice.

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in eukaryotic organisms and play essential roles in immunity and stress responses. However, the role of MAPKs in chloroplast development remains to be evidently established. RESULTS: In this study, a rice chlorosis seedling lethality 1 (csl1) mutant with a Zhonghua11 (ZH11, japonica) background was isolated. Seedlings of the mutant were characterized by chlorotic leaves and death after the trefoil stage, and chloroplasts were observed to contain accumulated starch granules. Molecular cloning revealed that OsCSL1 encoded a MAPK kinase kinase22 (MKKK22) targeted to the endoplasmic reticulum (ER), and functional complementation of OsCSL1 was found to restore the normal phenotype in csl1 plants. The CRISPR/Cas9 technology was used for targeted disruption of OsCSL1, and the OsCSL1-Cas9 lines obtained therein exhibited yellow seedlings which phenocopied the csl1 mutant. CSL1/MKKK22 was observed to establish direct interaction with MKK4, and altered expression of MKK1 and MKK4 was detected in the csl1 mutant. Additionally, disruption of OsCSL1 led to reduced expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded RNA polymerases, nuclear-encoded RNA polymerase, and nuclear-encoded chloroplast genes. CONCLUSIONS: The findings of this study revealed that OsCSL1 played roles in regulating the expression of multiple chloroplast synthesis-related genes, thereby affecting their functions, and leading to wide-ranging defects, including chlorotic seedlings and severely disrupted chloroplasts containing accumulated starch granules.

Karyotype diversity in cultivated and wild Indian rice through EMA-based chromosome analysis.

India is known for its diverse cultivated and wild rice germplasm. In today's crop improvement programmes, wild relatives are much-needed genetic repository of valuable traits. Analysis of genetic diversity at the chromosomal level is one cost-effective tool to unlock foundational information related to genetics and plant breeding. Presently, enzymatic maceration and air-drying method (EMA) has been applied for the first time in six cultivated and nine wild Indian rice (diploid and tetraploid). EMA method following Giemsa staining has yielded large numbers of cytoplasm free metaphase plates with distinct chromosome morphology. Detailed analysis has revealed karyotype diversities in terms of total chromatin length (TCL), chromosome morphology and location of sat chromosomes within and between the studied species. Most of the cultivated rice has gained additional amount in TCL during the period of domestication in comparison to their progenitor Oryza nivara. Morphological clarity of the small chromosomes of rice was much required and has helped to identify individual chromosomes in the diverse karyotypes. Diversity in landmark SAT chromosomes is another important observation, not reported previously in Indian rice. Present study has shown that in most of the O. sativa members, the 10th pair contains SAT except one where 6th pair is satellited. On the other hand, diversity of SAT in diploid and tetraplod wild species has been recorded on 5th, 7th and 8th chromosome pairs and on 9th, 12th, 22nd and 23rd chromosome pairs, respectively. Karyomorphometric indices has helped to construct dendrogram to elucidate intraspecies and interspecies relationships. Untapped genetic diversity recorded in Indian rice through chromosomal analysis will be useful to the breeders and genome researchers.

Microstructure investigation of plant architecture with X-ray microscopy.

In recent years, the plant morphology has been well studied by multiple approaches at cellular and subcellular levels. Two-dimensional (2D) microscopy techniques offer imaging of plant structures on a wide range of magnifications for researchers. However, subcellular imaging is still challenging in plant tissues like roots and seeds. Here we use a three-dimensional (3D) imaging technology based on the X-ray microscope (XRM) and analyze several plant tissues from different plant species. The XRM provides new insights into plant structures using non-destructive imaging at high-resolution and high contrast. We also utilized a workflow aiming to acquire accurate and high-quality images in the context of the whole specimen. Multiple plant samples including rice, tobacco, Arabidopsis and maize were used to display the differences of phenotypes. Our work indicates that the XRM is a powerful tool to investigate plant microstructure in high-resolution scale. Our work also provides evidence that evaluate and quantify tissue specific differences for a range of plant species. We also characterize novel plant tissue phenotypes by the XRM, such as seeds in Arabidopsis, and utilize them for novel observation measurement. Our work represents an evaluated spatial and temporal resolution solution on seed observation and screening.

Ginger Essential Oils-Loaded Nanoemulsions: Potential Strategy to Manage Bacterial Leaf Blight Disease and Enhanced Rice Yield.

The bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious rice diseases, causing huge yield losses worldwide. Several technologies and approaches have been opted to reduce the damage; however, these have had limited success. Recently, scientists have been focusing their efforts on developing efficient and environmentally friendly nanobactericides for controlling bacterial diseases in rice fields. In the present study, a scanning electron microscope (SEM), transmission electron microscope (TEM), and a confocal laser scanning microscope (CLSM) were utilized to investigate the mode of actions of ginger EOs on the cell structure of Xoo. The ginger EOs caused the cells to grow abnormally, resulting in an irregular form with hollow layers, whereas the dimethylsulfoxide (DMSO) treatment showed a typical rod shape for the Xoo cell. Ginger EOs restricted the growth and production of biofilms by reducing the number of biofilms generated as indicated by CLSM. Due to the instability, poor solubility, and durability of ginger EOs, a nanoemulsions approach was used, and a glasshouse trial was performed to assess their efficacy on BLB disease control. The in vitro antibacterial activity of the developed nanobactericides was promising at different concentration (50-125 µL/mL) tested. The efficacy was concentration-dependent. There was significant antibacterial activity recorded at higher concentrations. A glasshouse trial revealed that developed nanobactericides managed to suppress BLB disease severity effectively. Treatment at a concentration of 125 µL/mL was the best based on the suppression of disease severity index, AUDPC value, disease reduction (DR), and protection index (PI). Furthermore, findings on plant growth, physiological features, and yield parameters were significantly enhanced compared to the positive control treatment. In conclusion, the results indicated that ginger essential oils loaded-nanoemulsions are a promising alternative to synthetic antibiotics in suppressing Xoo growth, regulating the BLB disease, and enhancing rice yield under a glasshouse trial.

Rice SPL10 positively regulates trichome development through expression of HL6 and auxin-related genes.

Trichomes function in plant defenses against biotic and abiotic stresses; examination of glabrous lines, which lack trichomes, has revealed key aspects of trichome development and function. Tests of allelism in 51 glabrous rice (Oryza sativa) accessions collected worldwide identified OsSPL10 and OsWOX3B as regulators of trichome development in rice. Here, we report that OsSPL10 acts as a transcriptional regulator controlling trichome development. Haplotype and transient expression analyses revealed that variation in the approximately 700-bp OsSPL10 promoter region is the primary cause of the glabrous phenotype in the indica cultivar WD-17993. Disruption of OsSPL10 by genome editing decreased leaf trichome density and length in the NIL-HL6 background. Plants with genotype OsSPL10WD-17993 /HL6 generated by crossing WD-17993 with NIL-HL6 also had fewer trichomes in the glumes. HAIRY LEAF6 (HL6) encodes another transcription factor that regulates trichome initiation and elongation, and OsSPL10 directly binds to the HL6 promoter to regulate its expression. Moreover, the transcript levels of auxin-related genes, such as OsYUCCA5 and OsPIN-FORMED1b, were altered in OsSPL10 overexpression and RNAi transgenic lines. Feeding tests using locusts (Locusta migratoria) demonstrated that non-glandular trichomes affect feeding by this herbivore. Our findings provide a molecular framework for trichome development and an ecological perspective on trichome functions.

Tandem DNA repeats contain cis-regulatory sequences that activate biotrophy-specific expression of Magnaporthe effector gene PWL2.

During plant infection, fungi secrete effector proteins in coordination with distinct infection stages. Thus, the success of plant infection is determined by precise control of effector gene expression. We analysed the PWL2 effector gene of the rice blast fungus Magnaporthe oryzae to understand how effector genes are activated specifically during the early biotrophic stages of rice infection. Here, we used confocal live-cell imaging of M. oryzae transformants with various PWL2 promoter fragments fused to sensitive green fluorescent protein reporter genes to determine the expression patterns of PWL2 at the cellular level, together with quantitative reverse transcription PCR analyses at the tissue level. We found PWL2 expression was coupled with sequential biotrophic invasion of rice cells. PWL2 expression was induced in the appressorium upon penetration into a living rice cell but greatly declined in the highly branched hyphae when the first-invaded rice cell was dead. PWL2 expression then increased again as the hyphae penetrate into living adjacent cells. The expression of PWL2 required fungal penetration into living plant cells of either host rice or nonhost onion. Deletion and mutagenesis experiments further revealed that the tandem repeats in the PWL2 promoter contain 12-base pair sequences required for expression. We conclude that PWL2 expression is (a) activated by an unknown signal commonly present in living plant cells, (b) specific to biotrophic stages of fungal infection, and (c) requires 12-base pair cis-regulatory sequences in the promoter.

Roles of FERONIA-like receptor genes in regulating grain size and quality in rice.

Grain yield and quality are critical factors that determine the value of grain crops. In this study, we analyzed the functions of 12 FERONIA-like receptor (FLR) family members in rice and investigated their effects on grain size and quality. We found that FLR1, FLR2 and FLR8 negatively regulated grain size, and FLR15 positively regulated grain size. flr1 mutants had a higher cell number and an accelerated rate of grain filling compared to wild-type plants, which led to grains with greater widths. A mechanism underlying the regulation of grain size by FLR1 is that FLR1 is associated with OsRac1 Rho-like GTPase, a positive regulator of grain size. Regarding grain quality, the flr1 mutant had a higher percentage of chalkiness compared with wild-type plants, and seeds carrying mutations in flr3 and flr14 had endosperms with white floury cores. To elucidate the possible mechanism underlying this phenomenon, we found that FLR1 was constitutively expressed during endosperm development. RNA-seq analysis identified 2,367 genes that were differentially expressed in the flr1 mutant, including genes involved in starch and sucrose metabolism and carbon fixation. In this study, we identified the roles played by several FLR genes in regulating grain size and quality in rice and provided insights into the molecular mechanism governing the FLR1-mediated regulation of grain size.

Improvement of production yield and extraction efficacy of recombinant protein by high endosperm-specific expression along with simultaneous suppression of major seed storage proteins.

Human transforming growth factor-ß1 (hTGF-ß1)　was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins　(SSPs)　through RNA interference (RNAi). When driven by the 26 kDa α-globulin endosperm-specific promoter, it accumulated up to the markedly high level of 452 µg/grain. However, exchange with other seed-specific promoters such as 18 kDa oleosin and AGPase promoters resulted in remarkable reduction to the levels of 62 and 48 µg/grain, respectively, even though endogenous SSPs were reduced to the similar level. These production levels were almost similar to those (42 and 108 µg/grain) produced by the glutelin GluB-1 endosperm-specific promoter and the maize ubiquitin constitutive promoter without reduction of SSPs, respectively. When extracted from these transgenic rice seeds with reduced SSPs with various buffers, it could be solubilized with denaturant solution, which was in remarkable contrast with those without depressed SSPs which required further supplementation of reducing agent for extraction. This difference was associated with the fact that it was mainly deposited to ER-derived structures though self-aggregation or interaction with remaining prolamin via intermolecular disulfide bonds.

The Predicted Functional Compartmentation of Rice Terpenoid Metabolism by Trans-Prenyltransferase Structural Analysis, Expression and Localization.

Most terpenoids are derived from the basic terpene skeletons of geranyl pyrophosphate (GPP, C10), farnesyl-PP (FPP, C15) and geranylgeranyl-PP (GGPP, C20). The trans-prenyltransferases (PTs) mediate the sequential head-to-tail condensation of an isopentenyl-PP (C5) with allylic substrates. The in silico structural comparative analyses of rice trans-PTs with 136 plant trans-PT genes allowed twelve rice PTs to be identified as GGPS_LSU (OsGGPS1), homomeric G(G)PS (OsGPS) and GGPS_SSU-II (OsGRP) in Group I; two solanesyl-PP synthase (OsSPS2 and 3) and two polyprenyl-PP synthases (OsSPS1 and 4) in Group II; and five FPSs (OsFPS1, 2, 3, 4 and 5) in Group III. Additionally, several residues in "three floors" for the chain length and several essential domains for enzymatic activities specifically varied in rice, potentiating evolutionarily rice-specific biochemical functions of twelve trans-PTs. Moreover, expression profiling and localization patterns revealed their functional compartmentation in rice. Taken together, we propose the predicted topology-based working model of rice PTs with corresponding terpene metabolites: GPP/GGPPs mainly in plastoglobuli, SPPs in stroma, PPPs in cytosol, mitochondria and chloroplast and FPPs in cytosol. Our findings could be suitably applied to metabolic engineering for producing functional terpene metabolites in rice systems.

Grass-Specific EPAD1 Is Essential for Pollen Exine Patterning in Rice.

The highly variable and species-specific pollen surface patterns are formed by sporopollenin accumulation. The template for sporopollenin deposition and polymerization is the primexine that appears on the tetrad surface, but the mechanism(s) by which primexine guides exine patterning remain elusive. Here, we report that the Poaceae-specific EXINE PATTERN DESIGNER 1 (EPAD1), which encodes a nonspecific lipid transfer protein, is required for primexine integrity and pollen exine patterning in rice (Oryza sativa). Disruption of EPAD1 leads to abnormal exine pattern and complete male sterility, although sporopollenin biosynthesis is unaffected. EPAD1 is specifically expressed in male meiocytes, indicating that reproductive cells exert genetic control over exine patterning. EPAD1 possesses an N-terminal signal peptide and three redundant glycosylphosphatidylinositol (GPI)-anchor sites at its C terminus, segments required for its function and localization to the microspore plasma membrane. In vitro assays indicate that EPAD1 can bind phospholipids. We propose that plasma membrane lipids bound by EPAD1 may be involved in recruiting and arranging regulatory proteins in the primexine to drive correct exine deposition. Our results demonstrate that EPAD1 is a meiocyte-derived determinant that controls primexine patterning in rice, and its orthologs may play a conserved role in the formation of grass-specific exine pattern elements.

Anatomy of leaf apical hydathodes in four monocotyledon plants of economic and academic relevance.

Hydathode is a plant organ responsible for guttation in vascular plants, i.e. the release of droplets at leaf margin or surface. Because this organ connects the plant vasculature to the external environment, it is also a known entry site for several vascular pathogens. In this study, we present a detailed microscopic examination of leaf apical hydathodes in monocots for three crops (maize, rice and sugarcane) and the model plant Brachypodium distachyon. Our study highlights both similarities and specificities of those epithemal hydathodes. These observations will serve as a foundation for future studies on the physiology and the immunity of hydathodes in monocots.

GWC1 is essential for high grain quality in rice.

Appearance quality is an important determinant of rice quality. Many genes that affect grain appearance quality have been identified, but the regulatory mechanisms that contribute to this trait remain unclear. Here, two grains with chalkiness (gwc1) mutants, gwc1-1 and gwc1-2, were identified from an EMS-mutagenized population of indica rice cultivar Shuhui498 (R498). The gwc1 mutants had poor grain appearance quality consistent with the measured values for the percentage of grains with chalkiness, square of chalky endosperm, the total starch, amylose and sucrose contents. Milling quality and grain size were also affected in the gwc1 mutants. The gwc1-1 and gwc1-2 were found to be loss-of-function allelic mutants. GWC1 was mapped to the long arm of rice chromosome 8 using the MutMap strategy and incorrectly annotated in the reference genome for Nipponbare (MSU). The GWC1 gene corresponds to the WTG1/OsOTUB1 gene, which encodes an otubain-like protease with deubiquitinating activity that is homologous to human OTUB1. GWC1 transcripts accumulated to high levels in early endosperm after fertilization and developing inflorescences, and GWC1-green fluorescent protein (GFP) signal was detected in the nucleus and cytoplasm. GWC1 is likely to regulate grain appearance quality through genes involved in sucrose metabolism and starch biosynthesis. Overall, the present findings reveal that GWC1 is important for grain quality and yield due to its effects on grain chalkiness and size.

Microscopic and spectroscopic characterization of rice and corn starch.

Starch granules from rice and corn were isolated, and their molecular mechanism on interaction with α-amylase was characterized through biochemical test, microscopic imaging, and spectroscopic measurements. The micro-scale structure of starch granules were observed under an optical microscope and their average size was in the range 1-100 µm. The surface topological structures of starch with micro-holes due to the effect of α- amylase were also visualized under scanning electron microscope. The crystallinity was confirmed by X-ray diffraction patterns as well as second-harmonic generation microscopy. The change in chemical bonds before and after hydrolysis of the starch granules by α- amylase was determined by Fourier transform infrared spectroscopy. Combination of microscopy and spectroscopy techniques relates structural and chemical features that explain starch enzymatic hydrolysis which will provide a valid basis for future studies in food science and insights into the energy transformation dynamics.

OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways.

Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

Microstructure, gelatinization and pasting properties of rice starch under acid and heat treatments.

Microstructure of starch plays a vital role in the physicochemical properties. In this study, the microstructure, gelatinization and pasting properties of rice starches after acid, heat and acid-heat treatments were investigated. In our findings, molecular weight and size, amylopectin chain length distribution, degree of branching, amylose content, relative crystallinity, and hot water-solubility of rice starch were varied significantly after acid-heat treatment, showing a synergy between acid and heat. Correspondingly, acid-heat treatment markedly changed gelatinization and pasting properties of rice starch by sharply decreasing gelatinization and pasting temperatures as well as pasting viscosities. Pearson correlation analysis was performed, showing that molecular weight, size, amylopectin chain length distribution, and amylose content were significantly correlated to gelatinization temperatures, and crystallinity was remarkably correlated to gelatinization enthalpy. As for pasting properties, molecular weight and size as the major factors were substantially correlated to pasting viscosities and pasting temperature. This study provides a structure-property correlation for acid-heat treated rice starch, giving scientific basis for the application of starch in food industry.

Influence of germination time on the morphological, morphometric, structural, and physicochemical characteristics of Esmeralda and Perla barley starch.

The aim of this study was to analyzing the impact of germination time on the morphology, crystallinity, gelatinization and viscosity properties on the starch of Esmeralda and Perla barley variety. The two barley were germinated for 1 to 8 days, at 26 °C and 65% relative humidity. Micrographs showed the presence of pinholes and eroded surfaces. Starch in Esmeralda was hydrolyzed completely at 8 days of germination. Birefringence was reduced from day 4, losing molecular structuring of the crystalline area. Morphometric data: fractal dimension, area, perimeter, circularity, and roundness decreased significantly along germination time in both varieties. The entropy increased significantly, from 0.79 to 10.09 in Esmeralda and from 0.46 to 7.57 in Perla. Relative crystallinity decreased significantly in the Perla from 24.7% to 23.6%. Viscosity peaks were also significantly reduced, pasting temperature was constant in Esmeralda but in Perla was significantly reduced from 95.43 to 95.19 °C with germination, the gelatinization temperature increased significantly in the Esmeralda while in Perla it remained constant. Enthalpy decreased significantly to 75.8% and 37% in Esmeralda and Perla respectively. The study of germination impact on structural and physicochemical properties is important to identify the use of hydrolyzed starches in the food industry or others.

A point mutation in LTT1 enhances cold tolerance at the booting stage in rice.

The cold tolerance of rice at the booting stage is a main factor determining sustainability and regional adaptability. However, relatively few cold tolerance genes have been identified that can be effectively used in breeding programmes. Here, we show that a point mutation in the low-temperature tolerance 1 (LTT1) gene improves cold tolerance by maintaining tapetum degradation and pollen development, by activation of systems that metabolize reactive oxygen species (ROS). Cold-induced ROS accumulation is therefore prevented in the anthers of the ltt1 mutants allowing correct development. In contrast, exposure to cold stress dramatically increases ROS accumulation in the wild type anthers, together with the expression of genes encoding proteins associated with programmed cell death and with the accelerated degradation of the tapetum that ultimately leads to pollen abortion. These results demonstrate that appropriate ROS management is critical for the cold tolerance of rice at the booting stage. Hence, the ltt1 mutation can significantly improve the seed setting ability of cold-sensitive rice varieties under low-temperature stress conditions, with little yield penalty under optimal temperature conditions. This study highlights the importance of a valuable genetic resource that may be applied in rice breeding programmes to enhance cold tolerance.

Multilayered Regulation of Membrane-Bound ONAC054 Is Essential for Abscisic Acid-Induced Leaf Senescence in Rice.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054ß), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054ß lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

WHITE STRIPE LEAF8, encoding a deoxyribonucleoside kinase, is involved in chloroplast development in rice.

KEY MESSAGE: WSL8 encoding a deoxyribonucleoside kinase (dNK) that catalyzes the first step in the salvage pathway of nucleotide synthesis plays an important role in early chloroplast development in rice. The chloroplast is an organelle that converts light energy into chemical energy; therefore, the normal differentiation and development of chloroplast are pivotal for plant survival. Deoxyribonucleoside kinases (dNKs) play an important role in the salvage pathway of nucleotides. However, the relationship between dNKs and chloroplast development remains elusive. Here, we identified a white stripe leaf 8 (wsl8) mutant that exhibited a white stripe leaf phenotype at seedling stage (before the four-leaf stage). The mutant showed a significantly lower chlorophyll content and defective chloroplast morphology, whereas higher reactive oxygen species than the wild type. As the leaf developed, the chlorotic mutant plants gradually turned green, accompanied by the restoration in chlorophyll accumulation and chloroplast ultrastructure. Map-based cloning revealed that WSL8 encodes a dNK on chromosome 5. Compared with the wild type, a C-to-G single base substitution occurred in the wsl8 mutant, which caused a missense mutation (Leu 349 Val) and significantly reduced dNK enzyme activity. A subcellular localization experiment showed the WSL8 protein was targeted in the chloroplast and its transcripts were expressed in various tissues, with more abundance in young leaves and nodes. Ribosome and RNA-sequencing analysis indicated that some components and genes related to ribosome biosynthesis were down-regulated in the mutant. An exogenous feeding experiment suggested that the WSL8 performed the enzymic activity of thymidine kinase, especially functioning in the salvage synthesis of thymidine monophosphate. Our results highlight that the salvage pathway mediated by the dNK is essential for early chloroplast development in rice.