Salicylate and glutamate mediate different Cd accumulation and tolerance between Brassica napus and B. juncea.

Most hyperaccumulator plants have little economic values, and therefore have not been widely used in Cd-contaminated soils. Rape species are Cd hyperaccumulators with high economic values. Black mustard seed (Brassica juncea) has a higher accumulation ability and a higher tolerance for Cd than oilseed rape (Brassica napus), but its biomass is relatively low and its geographical distribution is limited. However, it is unknown why B. juncea (Bj) is more tolerant to and accumulates more Cd than B. napus (Bn). Here, we found that the differences in Cd accumulation and tolerance between the two species is mainly because Bj plants have higher levels of salicylic acid and glutamic acid than Bn plants. Exogenous salicylate and glutamate treatments enhanced Cd accumulation (salicylate + glutamate co-treatment doubled Cd accumulation level in Bn seedlings) but reduced oxidative stresses by increasing glutathione biosynthesis and activating phytochelatin-based sequestration of Cd into vacuoles. Our results provide a new idea to simultaneously improve Cd accumulation and Cd tolerance in B. napus.

The false smut pathogen Ustilaginoidea virens requires rice stamens for false smut ball formation.

Rice false smut has emerged as a serious grain disease in rice production worldwide. The disease is characterized by the transformation of individual rice florets into false smut balls, which is caused by the fungal pathogen Ustilaginoidea virens. To date, little is known about the host factors required for false smut ball formation by U. virens. In this study, we identified histological determinants for the formation of false smut balls by inoculating U. virens into rice floral mutants defective with respect to individual floral parts. The results showed that U. virens could form mature false smut balls in rice floral mutants with defective pistils, but failed to develop false smut balls in the superwoman mutant lacking stamens, identifying that U. virens requires rice stamens to complete its infection cycle. Comparative transcriptome analysis indicated a list of candidate host genes that may facilitate nutrient acquisition by U. virens from the rice stamens, such as SWEET11, SWEET14 and SUT5, and genes involved in the biosynthesis of trehalose and raffinose family sugars. These data pinpoint rice stamens as the key target organ of U. virens infection and provide a valuable starting point for dissecting the molecular mechanism of false smut ball formation.

Two-factor ANOVA of SSH and RNA-seq analysis reveal development-associated Pi-starvation genes in oilseed rape.

MAIN CONCLUSION: The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.

Parental Genome Imbalance Causes Post-Zygotic Seed Lethality and Deregulates Imprinting in Rice.

BACKGROUND: Reproductive isolation between rice of different ploidy levels is manifested as endosperm and embryo abortion in seeds produced by interploidy crosses. Genomic imprinting is considered to be the underlying mechanism establishing the post-zygotic hybridization barrier. We characterized disrupted seed development in reciprocal crosses between a diploid Japonica rice and a tetraploid Indica rice. RESULTS: Triploid seeds from these crosses had aborted development and could not germinate in soil but could be rescued in culture medium with significantly more seeds developing to seedlings in the 4n × 2n (♀-♂) cross with excess maternal genomes than in the 2n × 4n cross with excess paternal genome. Consistent with previous findings, precocious endosperm cellularization and bigger embryos were observed in the seeds from the maternal excess cross, whereas absence of cellularization and arrested globular embryos were found in the seeds from the paternal excess cross, supporting the idea that endosperm cellularization is an important transition for embryo development. Moreover, we found that starch granules were persistently deposited in the pericarp parenchyma cells of the paternal excess cross, while pericarp starch gradually decreased and relocated to the developing endosperm in balanced and maternal excess crosses in which cellularization and starch deposition occur in endosperm, suggesting that parental genome balance influences pericarp starch relocation via cellularization and starch deposition. Loss of imprinting, or altered expression of imprinted genes and epigenetic regulators, OsFIE2 and OsMET1b were observed, implying the potential role of imprinting and epigenetic mechanisms in regulating the differential parental genome dosage effects on endosperm development. CONCLUSIONS: Our results support the hypothesis that the maternal genome dosage promotes endosperm cellularization and the paternal genome dosage delays or inhibits cellularization via contributing different sets of imprinted genes.

Global Methylation Patterns and Their Relationship with Gene Expression and Small RNA in Rice Lines with Different Ploidy.

Whole genome duplication (WGD) is a major force in angiosperm evolution. Whether WGD is accompanied by the evolution of epigenetic regulators remains to be explored. Here we investigate whole genome methylation, gene expression, and miRNA regulation among monoploid, diploid, and triploid rice plants isolated from a twin-seedling population. The DNA methylation patterns in the three different ploidy plants were highly similar, with DNA methylation primarily enriched in the promoters. We examined the methylation of single genes and detected around 25,500 methylated genes, of which 22,751 were methylated in all three lines. Significantly divergent DNA methylation patterns between each pair of three lines were only detected in 64 genes, though more genes were found to exhibit differential expression. Analysis of DNA methylation and expression patterns showed that higher DNA methylation levels upstream of the transcription start sites are correlated with higher levels of expression of related genes; whereas higher DNA methylation levels in gene body regions are correlated with lower levels of expression. We also carried out high-throughput sequencing of small RNA libraries and identified 36 new miRNAs. These miRNAs have different expression levels depending on the ploidy.

Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae.

MicroRNAs (miRNAs) are indispensable regulators for development and defense in eukaryotes. However, the miRNA species have not been explored for rice (Oryza sativa) immunity against the blast fungus Magnaporthe oryzae, the most devastating fungal pathogen in rice production worldwide. Here, by deep sequencing small RNA libraries from susceptible and resistant lines in normal conditions and upon M. oryzae infection, we identified a group of known rice miRNAs that were differentially expressed upon M. oryzae infection. They were further classified into three classes based on their expression patterns in the susceptible japonica line Lijiangxin Tuan Hegu and in the resistant line International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake that contains a single resistance gene locus, Pyricularia-Kanto 51-m (Pikm), within the Lijiangxin Tuan Hegu background. RNA-blot assay of nine of them confirmed sequencing results. Real-time reverse transcription-polymerase chain reaction assay showed that the expression of some target genes was negatively correlated with the expression of miRNAs. Moreover, transgenic rice plants overexpressing miR160a and miR398b displayed enhanced resistance to M. oryzae, as demonstrated by decreased fungal growth, increased hydrogen peroxide accumulation at the infection site, and up-regulated expression of defense-related genes. Taken together, our data indicate that miRNAs are involved in rice immunity against M. oryzae and that overexpression of miR160a or miR398b can enhance rice resistance to the disease.

[Imprinting genes and it's expression in Arabidopsis].

Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.

[cDNA-AFLP analysis among different organs of haploid and diploid plants in twin-seedling rice].

SARII-628 is one of the natural twin-seedling populations, which are maintained in Rice Institution of Sichuan Agricultural University. Haploid-diploid (n: 2n) twin-seedlings can annually emerge from SARII-628. In this study, cDNA-AFLP technique was applied for these haploid and diploid plants. The double stranded cDNA were synthesized from the total RNAs of the roots, stems, panicles, and leaves of three haploid and one diploid plants. Then, EcoRI/MspI combination was chosen for expression difference analysis with cDNA-AFLP technique among the 16 samples. Thirty pairs of primers were selected to amplify 633 loci. Among them, 49 loci were differently expressed. As shown by cDNA-AFLP, the expression levels of haploids were different among individuals but not significant. The average ratio of sensitive loci (activated loci + silenced loci) in haploids was 5.14%, which was higher than that of diploid (3.93%). According to the presence or absence of the bands in root, stem, panicle, and leaf, out of the 14 types of amplified patterns, only 4 exclusively occurred in haploid and the corresponding loci were thus haploid-specific in expression. The collecting analysis for the silenced and activated loci among different organs of the three haploids revealed that the silenced loci were more than the activated ones in roots and panicles. An opposite trend was observed in panicles and leaves. However, the total number of the activated loci in all the four types of haploid organs was more than that of the silenced loci. The number of sensitive loci is the highest in root, while that in panicle is the least, which suggested that gene expressions were organ-specific.

Phenotypic characterization, genetic analysis and gene-mapping for a brittle mutant in rice.

Plant mechanical strength is an important agronomic trait of rice. An ethyl methane sulfonate (EMS)-induced rice mutant, fragile plant 2 (fp2), showed morphological changes and reduced mechanical strength. Genetic analysis indicated that the brittle of fp2 was controlled by a recessive gene. The fp2 gene was mapped on chromosome 10. Anatomical analyses showed that the fp2 mutation caused the reduction of cell length and cell wall thickness, increasing of cell width, and the alteration of cell wall structure as well as the vessel elements. The consequence was a global alteration in plant morphology. Chemical analyses indicated that the contents of cellulose and lignin decreased, and hemicelluloses and silicon increased in fp2. These results were different from the other mutants reported in rice. Thus, fp2 might affect the deposition and patterning of microfibrils, the biosynthesis and deposition of cell wall components, which influences the formation of primary and secondary cell walls, the thickness of cell walls, cell elongation and expansion, plant morphology and plant strength in rice.

[Embryological observation on the wide cross of rice and maize].

In this report, we studied the fertilization and embryo development of a cross between rice and maize. The results showed that the pollen grains of maize could enter into the tissue of style of rice, and reached the embryo sac and released the sperms. Maize sperm might fuse with rice egg or polar nucleus and formed zygote or primary endosperm. But the embryo and endosperm could not be developed normally, and thus the underdeveloped hybrid seeds were formed. The reason was probably that the parental genomes could not coordinate well. This work provided a clue to solve the crossing obstacles between different species.

Genome-wide high-frequency non-Mendelian loss of heterozygosity in rice.

Classic Mendelian genetics declares that hybrids inherit genomic information from both male and female parents, and that alleles should be heterozygous in F1 plants. A few exceptions to this principle have been reported, but most of them are restricted to either a limited set of specific genes or specific types of alleles. Here, we show that a rice triploid and diploid hybridization resulted in stable diploid progenies, both in genotypes and phenotypes, through gene homozygosity. Furthermore, their gene homozygosity can be inherited through 8 generations, and they can convert DNA sequences of other rice varieties into their own. Molecular-marker examination confirmed that this type of genome-wide gene conversion occurred at a very high frequency. Possible mechanisms, including RNA-templated repair of double-strand DNA, are discussed.

[Cytological and embryological observation on the cross of autotriploid x diploid in rice].

In this report, the process of hybridization between different ploidy levels was observed in rice. Autotriploid (2n = 36) 149-B, generated naturally from the twin-seedling rice population SAR-2, was used as the female parent crossing with a normal diploid variety Shuhui 363. The processes of fertilization and embryological development were investigated. The pollen could germinate in triploid tissue of pistil normally and arrive at micropyle. But the percentage of double fertilization was only 8.89%, and the percentage of seed setting was 0.566%. The hybridization obstacles mainly occurred in the process of fertilization. Barriers of double fertilization, prolongation and abnormal development of proembryo, and even development stopping or differentiation failure of embryo and/or endosperm could result in abnormal development and abortion of zygote.

Genetic analysis and histological study of red seed in rice.

Reciprocal crosses between red and achromatic rice revealed that the seed color of F1 was determined by its female parent. According to the seed color and plant segregation ratio of F1, F2, and F3 generations, the red phenotype of red double-haploid seed was determined by a dominant, monogene with maternal effect. Histological study showed that the red pigments accumulated in the pericarp layer only. The assay of developmental timing of pigment accumulation showed that the red color accumulated from desiccation stage to perfectly maturation stage of the seeds.