OsMADS1 Regulates Grain Quality, Gene Expressions, and Regulatory Networks of Starch and Storage Protein Metabolisms in Rice.

OsMADS1 plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated OsMADS1Olr allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1Olr-eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of OsMADS1 also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that OsMADS1 regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of OsMADS1 in regulating rice grain quality but also highlight the application potentials of OsMADS1 and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

Cell Wall Matrix Polysaccharides Contribute to Salt-Alkali Tolerance in Rice.

Salt-alkali stress threatens the resilience to variable environments and thus the grain yield of rice. However, how rice responds to salt-alkali stress at the molecular level is poorly understood. Here, we report isolation of a novel salt-alkali-tolerant rice (SATR) by screening more than 700 germplasm accessions. Using 93-11, a widely grown cultivar, as a control, we characterized SATR in response to strong salt-alkali stress (SSAS). SATR exhibited SSAS tolerance higher than 93-11, as indicated by a higher survival rate, associated with higher peroxidase activity and total soluble sugar content but lower malonaldehyde accumulation. A transcriptome study showed that cell wall biogenesis-related pathways were most significantly enriched in SATR relative to 93-11 upon SSAS. Furthermore, higher induction of gene expression in the cell wall matrix polysaccharide biosynthesis pathway, coupled with higher accumulations of hemicellulose and pectin as well as measurable physio-biochemical adaptive responses, may explain the strong SSAS tolerance in SATR. We mapped SSAS tolerance to five genomic regions in which 35 genes were candidates potentially governing SSAS tolerance. The 1,4-ß-D-xylan synthase gene OsCSLD4 in hemicellulose biosynthesis pathway was investigated in details. The OsCSLD4 function-disrupted mutant displayed reduced SSAS tolerance, biomass and grain yield, whereas the OsCSLD4 overexpression lines exhibited increased SSAS tolerance. Collectively, this study not only reveals the potential role of cell wall matrix polysaccharides in mediating SSAS tolerance, but also highlights applicable value of OsCSLD4 and the large-scale screening system in developing SSAS-tolerant rice.

Dual RNA Sequencing Reveals the Genome-Wide Expression Profiles During the Compatible and Incompatible Interactions Between Solanum tuberosum and Phytophthora infestans.

Late blight, caused by Phytophthora infestans (P. infestans), is a devastating plant disease. P. infestans genome encodes hundreds of effectors, complicating the interaction between the pathogen and its host and making it difficult to understand the interaction mechanisms. In this study, the late blight-resistant potato cultivar Ziyun No.1 and the susceptible potato cultivar Favorita were infected with P. infestans isolate SCPZ16-3-1 to investigate the global expression profiles during the compatible and incompatible interactions using dual RNA sequencing (RNA-seq). Most of the expressed Arg-X-Leu-Arg (RXLR) effector genes were suppressed during the first 24 h of infection, but upregulated after 24 h. Moreover, P. infestans induced more specifically expressed genes (SEGs), including RXLR effectors and cell wall-degrading enzymes (CWDEs)-encoding genes, in the compatible interaction. The resistant potato activated a set of biotic stimulus responses and phenylpropanoid biosynthesis SEGs, including kirola-like protein, nucleotide-binding site-leucine-rich repeat (NBS-LRR), disease resistance, and kinase genes. Conversely, the susceptible potato cultivar upregulated more kinase, pathogenesis-related genes than the resistant cultivar. This study is the first study to characterize the compatible and incompatible interactions between P. infestans and different potato cultivars and provides the genome-wide expression profiles for RXLR effector, CWDEs, NBS-LRR protein, and kinase-encoding genes.

Characterization of the 'Oat-Like Rice' Caused by a Novel Allele OsMADS1Olr Reveals Vital Importance of OsMADS1 in Regulating Grain Shape in Oryza sativa L.

BACKGROUND: Grain shape is a critical agronomic trait affecting grain yield and quality. Exploration and functional characterization of grain shape-related genes will facilitate rice breeding for higher quality and yield. RESULTS: Here, we characterized a recessive mutant named Oat-like rice for its unique grain shape which highly resembles oat grains. The Oat-like rice displayed abnormal floral organs, an open hull formed by remarkably elongated leafy lemmas and paleae, occasionally formed conjugated twin brown rice, an aberrant grain shape and a low seed setting rate. By map-based cloning, we discovered that Oat-like rice harbors a novel allele of OsMADS1 gene (OsMADS1Olr), which has a spontaneous point mutation that causes the substitution of an amino acid that is highly conserved in the MADS-box domain of the MADS-box family. Further linkage analysis indicated that the point mutation in the OsMADS1Olr is associated with Oat-like rice phenotype, and expression analysis of the OsMADS1 by qRT-PCR and GUS staining also indicated that it is highly expressed in flower organs as well as in the early stages of grain development. Furthermore, OsMADS1Olr-overexpressing plants showed similar phenotypes of Oat-like rice in grain shape, possibly due to the dominant negative effect. And OsMADS1-RNAi plants also displayed grain phenotypes like Oat-like rice. These results suggested that OsMADS1Olr is responsible for the Oat-like rice phenotype including aberrant grain shape. Moreover, the expression levels of representative genes related to grain shape regulation were apparently altered in Oat-like rice, OsMADS1Olr-overexpressing and OsMADS1-RNAi transgenic plants. Finally, compared with Oat-like rice, OsMADS1Olr-overexpressing and OsMADS1-RNAi plants, mild phenotype of seed-specific OsMADS1-RNAi transgenic plants indicated that OsMADS1 may has has a direct regulation role in grain development and the grain phenotypes of Oat-like rice, OsMADS1Olr-overexpressing and OsMADS1-RNAi plants are majorly caused by the abnormal lemma and palea development. CONCLUSIONS: Altogether, our results showed that grain shape and a low seed setting rate of the notable 'Oat-like rice' are caused by a spontaneous point mutation in the novel allele OsMADS1Olr. Furthermore, our findings suggested that OsMADS1 mediates grain shape possibly by affecting the expression of representative genes related to grain shape regulation. Thus, this study not only revealed that OsMADS1 plays a vital role in regulating grain shape of rice but also highlighted the importance and value of OsMADS1 to improve the quality and yield of rice by molecular breeding.

Proteomic Analysis of Rice Subjected to Low Light Stress and Overexpression of OsGAPB Increases the Stress Tolerance.

BACKGROUND: Light provides the energy for photosynthesis and determines plant morphogenesis and development. Low light compromises photosynthetic efficiency and leads to crop yield loss. It remains unknown how rice responds to low light stress at a proteomic level. RESULTS: In this study, the quantitative proteomic analysis with isobaric tags for relative and absolute quantitation (iTRAQ) was used and 1221 differentially expressed proteins (DEPs) were identified from wild type rice plants grown in control or low light condition (17% light intensity of control), respectively. Bioinformatic analysis of DEPs indicated low light remarkably affects the abundance of chloroplastic proteins. Specifically, the proteins involved in carbon fixation (Calvin cycle), electron transport, and ATPase complex are severely downregulated under low light. Furthermore, overexpression of the downregulated gene encoding rice ß subunit of glyceraldehyde-3-phosphate dehydrogenase (OsGAPB), an enzyme in Calvin cycle, significantly increased the CO2 assimilation rate, chlorophyll content and fresh weight under low light conditions but have no obvious effect on rice growth and development under control light. CONCLUSION: Our results revealed that low light stress on vegetative stage of rice inhibits photosynthesis possibly by decreasing the photosynthetic proteins and OsGAPB gene is a good candidate for manipulating rice tolerance to low light stress.

The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress-induced accumulation of ABA and proline.

Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, is required for many physiological reactions and processes. However, it remains largely unknown how NAD affects plant response to salt stress. We isolated a salt-sensitive mutant named hypersensitive to salt stress (hss) from an ethyl methanesulfonate-induced mutation population. A point mutation was identified by MutMap in the encoding region of Quinolinate Synthase (QS) gene required for the de novo synthesis of NAD. This point mutation caused a substitution of amino acid in the highly-conserved NadA domain of QS, resulting in an impairment of NAD biosynthesis in the mutant. Molecular and chemical complementation have restored the response of the hss mutant to salt stress, indicating that the decreased NAD contents in the mutant were responsible for its hypersensitivity to salt stress. Furthermore, the endogenous levels of abscisic acid (ABA) and proline were also reduced in stress-treated hss mutant. The application of ABA or proline could alleviate stress-induced oxidative damage of the mutant and partially rescue its hypersensitivity to salt stress, but not affect NAD concentration. Taken together, our results demonstrated that the NadA domain of QS is important for NAD biosynthesis, and NAD participates in plant response to salt stress by affecting stress-induced accumulation of ABA and proline.

SlNAC1, a stress-related transcription factor, is fine-tuned on both the transcriptional and the post-translational level.

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.

RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.).

BACKGROUND: Aromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (fgr/OsBADH2) homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of OsBADH2 is lacking. To elucidate the physiological roles of OsBADH2, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of OsBADH2 gene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of OsBADH2 and the fragrant compound in wild type and transgenic OsBADH2-RNAi repression lines, respectively. RESULTS: The results showed that multiple mutations identical to fgr allele occur in the 13 fragrant rice accessions across China; OsBADH2 is expressed constitutively, with less expression abundance in mature roots; the disrupted OsBADH2 by RNA interference leads to significantly increased 2-acetyl-1-pyrroline production. CONCLUSION: We have found that the altered expression levels of OsBADH2 gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin.

Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays).

Aldehyde dehydrogenases (ALDHs) play a central role in detoxification processes of aldehydes generated in plants when exposed to the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Zea mays, an ALDH (ZmALDH22A1) gene was isolated and characterized. ZmALDH22A1 belongs to the family ALDH22 that is currently known only in plants. The ZmALDH22A1 encodes a protein of 593 amino acids that shares high identity with the orthologs from Saccharum officinarum (95%), Oryza sativa (89%), Triticum aestivum (87%) and Arabidopsis thaliana (77%), respectively. Real-time PCR analysis indicates that ZmALDH22A1 is expressed differentially in different tissues. Various elevated levels of ZmALDH22A1 expression have been detected when the seedling roots exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Tomato stable transformation of construct expressing the ZmALDH22A1 signal peptide fused with yellow fluorescent protein (YFP) driven by the CaMV35S-promoter reveals that the fusion protein is targeted to plastid. Transgenic tobacco plants overexpressing ZmALDH22A1 shows elevated stresses tolerance. Stresses tolerance in transgenic plants is accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation.

An unusual posttranscriptional processing in two betaine aldehyde dehydrogenase loci of cereal crops directed by short, direct repeats in response to stress conditions.

Various abilities to synthesize and accumulate glycine betaine (GB) are crucial for angiosperms to develop salt and drought tolerances. In higher plants, GB is synthesized by a two-step oxidation of choline via an intermediate form of betaine aldehyde, and catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). In this study, numerous truncated and/or recombinant transcripts of two BADH homologs resulting from an unusual posttranscriptional processing were detected in rice (Oryza sativa) and other cereal crops, including maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare). The observed events took place at the 5' exonic region, and led to the insertion of exogenous gene sequences and a variety of deletions that resulted in the removal of translation initiation codon, loss of functional domain, and frame-shifts with premature termination by introducing stop codon. By contrast, the BADH transcripts from dicotyledonous species, such as spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum), had correctly processed mRNA. This suggests the differentiation of posttranscriptional processing in BADH genes potentially contributes to the variation of GB-synthesizing capacities among various plant species. In addition, comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short, direct repeats) are of paired presence surrounding the junctions of both the deletion and/or insertion sites in the unusual BADH transcripts. The site selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of short, direct repeats are probably required for the recognition of the deletion/insertion sites.