An ATP-binding cassette transporter, OsABCB24, is involved in female gametophyte development and early seed growth in rice.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP-binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%-100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two-nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane-localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5- to 2-fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

Dry-milled flour rice 'Seolgaeng' harbors a mutated fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase2.

'Seolgaeng', an opaque-endosperm rice (Oryza sativa) mutant, is used to prepare high-quality dry-milled rice flour. The mutation causing its opaque-endosperm phenotype was unknown. Map-based cloning identified a missense mutation in the gene FRUCTOSE-6-PHOSPHATE 2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE 2 (OsF2KP2) in Seolgaeng. Transfer DNA insertion and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced f2kp2 mutants exhibited opaque endosperm. Rice harbors another F2KP gene, OsF2KP1. CRISPR/Cas9-induced double mutants of OsF2KP1 and OsF2KP2 (f2kp-d) possessed more opaque endosperm compared to f2kp2 single mutants, whereas the endosperm of the f2kp1 single mutant was normal. Grain hardness and damaged starch content were significantly reduced in f2kp2 mutants compared to the wild type and f2kp1. Amylose content was lower than normal in f2kp2 mutants but not f2kp1. Grain hardness and amylose content were much lower in f2kp-d than in f2kp2. Starch polymerization analysis revealed altered amylopectin structure in f2kp2 and f2kp-d mutants. F2KP activity was lower in f2kp2 and much lower in the double mutants when compared to the wild types, but f2kp1 showed no significant difference. In coleoptiles, hypoxia induced OsF2KP2 expression but downregulated OsF2KP1. These results suggest that OsF2KP2 functions as the main F2KP isoform in endosperm experiencing hypoxia, but OsF2KP1 may partially compensate for the absence of OsF2KP2. We propose that F2KP has a crucial role in inorganic pyrophosphate-utilizing energy metabolism for starch biosynthesis in rice endosperm.

CRISPR/Cas-mediated editing of cis-regulatory elements for crop improvement.

To improve future agricultural production, major technological advances are required to increase crop production and yield. Targeting the coding region of genes via the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has been well established and has enabled the rapid generation of transgene-free plants, which can lead to crop improvement. The emergence of the CRISPR/Cas system has also enabled scientists to achieve cis-regulatory element (CRE) editing and, consequently, engineering endogenous critical CREs to modulate the expression of target genes. Recent genome-wide association studies have identified the domestication of natural CRE variants to regulate complex agronomic quantitative traits and have allowed for their engineering via the CRISPR/Cas system. Although engineering plant CREs can be advantageous to drive gene expression, there are still many limitations to its practical application. Here, we review the current progress in CRE editing and propose future strategies to effectively target CREs for transcriptional regulation for crop improvement.

Rice ß-glucosidase Os12BGlu38 is required for synthesis of intine cell wall and pollen fertility.

Glycoside hydrolase family1 ß-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice ß-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited ß-glucosidase activity on the universal substrate p-nitrophenyl ß-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated ß-glucosidase is necessary for proper intine development.

Action of Multiple Rice ß-Glucosidases on Abscisic Acid Glucose Ester.

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of ß-glucosidases. Evaluation of previously characterized recombinant rice ß-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher ß-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The ß-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular ß-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.

Loss of Function of Rice Plastidic Glycolate/Glycerate Translocator 1 Impairs Photorespiration and Plant Growth.

Ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic carbon fixation, is able to accept both O2 and CO2 as substrates. When it fixes O2, it produces 2-phosphoglycolate, which is detoxified by photorespiration and recycled to the Calvin-Benson-Bassham cycle. To complete photorespiration, metabolite transport across three organelles, chloroplasts, peroxisomes, and mitochondria, is necessary through transmembrane transporters. In rice (Oryza sativa) little is known about photorespiratory transmembrane transporters. Here, we identified the rice plastidic glycolate/glycerate translocator 1 (OsPLGG1), a homolog of Arabidopsis PLGG1. OsPLGG1 mutant lines, osplgg1-1, osplgg1-2, and osplgg1-3, showed a growth retardation phenotype, such as pale green leaf, reduced tiller number, and reduced seed grain weight as well as reduced photosynthetic carbon reduction rate due to low activities of photosystem I and II. The plant growth retardation in osplgg1 mutants was rescued under high CO2 condition. Subcellular localization of OsPLGG1-GFP fusion protein, along with its predicted N-terminal transmembrane domain, confirmed that OsPLGG1 is a chloroplast transmembrane protein. Metabolite analysis indicated significant accumulation of photorespiratory metabolites, especially glycolate and glycerate, which have been shown to be transported by the Arabidopsis PLGG1, and changes for a number of metabolites which are not intermediates of photorespiration in the mutants. These results suggest that OsPLGG1 is the functional plastidic glycolate/glycerate transporter, which is necessary for photorespiration and growth in rice.