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The Alfin-like (AL) family is a group of small plant-specific transcriptional factors involved in abiotic stresses in dicotyledon. In an early study, we found an AL gene in rice that was associated with grain yield under drought stress. However, little information is known about the AL family in rice. In this study, AL genes in the rice genome were identified, and the OsAL proteins were found to locate in the nucleus and have no transcriptional self-activation activity. The expression of the OsALs was regulated by different environmental stimulations and plant hormones. Association and domestication analysis revealed that natural variation of most OsALs was significantly associated with yield traits, drought resistance and divergence in grain size in indica and japonica rice varieties. Hap1 of OsAL7.1 and Hap7 of OsAL11 were favorable haplotypes of seed weight and germination under osmotic stress. Furthermore, osal7.1 and osal11 mutants have larger seeds and are more sensitive to abscisic acid and mannitol during germination stage. Overexpressing of OsAL7.1 and OsAL11 in rice weakened the tolerance to drought in the adult stage. Thus, our work provides informative knowledge for exploring and harnessing haplotype diversity of OsALs to improve yield stability under drought stress.

Mosses are one of the earliest diverging land plants that adapted to living on land. The BURP domain-containing proteins (BURP proteins) are plant-specific proteins that appeared when plants shifted from aquatic environments to land. Phylogenetic analysis revealed that the BURP domain of higher plants is originated from lower land plants and divergent because of motif conversion. To discover the function of BURP protein in moss, rice transgenics with ectopic expression of PpBURP2 were subjected to different abiotic stresses treatments. The results revealed that the ectopic expression of PpBURP2 enhanced the tolerance to osmotic and saline stresses at the seedling stage and drought stress at the adult stage. Further ectopic expression of PpBURP2 improved the cadmium (2+) (Cd2+) tolerance and reduced Cd2+ accumulation in rice leaves. Transcriptomic analysis of the transgenic PpBURP2 plants showed that the differentially expressed genes were involved in the metabolism of secondary metabolites, energy, oxidation-reduction process, and defense-related genes. Further experiments showed that the photosynthetic efficiency and resistance against bacterial leaf blight were obviously improved in transgenic plants. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed the physical interaction of BURP domain protein from rice and moss with mitogen-activated protein kinase kinase (MKK) from rice. Therefore, our findings demonstrate that overexpressing PpBURP2 in rice confers resistance to abiotic stresses and bacterial leaf blight. They also suggested that the regulatory role of BURP-like proteins across lower and higher plants was evolutionary conservation of responses of different classes of plants to different environmental challenges.

Drought stress adversely affects crop growth and productivity worldwide. In response, plants have evolved several strategies in which numerous genes are induced to counter stress. High mobility group (HMG) proteins are the second most abundant family of chromosomal proteins. They play a crucial role in gene transcriptional regulation by modulating the chromatin/DNA structure. In this study, we isolated a novel HMG gene, OsHMGB707, one of the candidate genes localized in the quantitative trait loci (QTL) interval of rice drought tolerance, and examined its function on rice stress tolerance. The expression of OsHMGB707 was up-regulated by dehydration and high salt treatment. Its overexpression significantly enhanced drought tolerance in transgenic rice plants, whereas its knockdown through RNA interference (RNAi) did not affect the drought tolerance of the transgenic rice plants. Notably, OsHMGB707-GFP is localized in the cell nucleus, and OsHMGB707 is protein-bound to the synthetic four-way junction DNA. Several genes were up-regulated in OsHMGB707-overexpression (OE) rice lines compared to the wild-type rice varieties. Some of the genes encode stress-related proteins (e.g., DREB transcription factors, heat shock protein 20, and heat shock protein DnaJ). In summary, OsHMGB707 encodes a stress-responsive high mobility group protein and regulates rice drought tolerance by promoting the expression of stress-related genes.

OsNAC45 belongs to the NAC family of (for NAM/ATAF1/2/CUC2) transcription factors, which play an important role in plant development and abiotic stress responses. Transgenic rice plants overexpressing OsNAC45 have exhibited improved tolerance to salt and drought stress. This study aimed to investigate the mechanism underlying the effects of OsNAC45 on root development under abiotic stress in rice Zhonghua11 (ZH11) (Oryza sativa L.). The OsNAC45 GUS expression was strong in roots of the adult stage, weak in the seedling stage, and induced by low-temperature stress in the seedling stage. Under NaCl and low temperature stress, the OsNAC45 overexpression (OE) lines exhibited maintenance of root structure and normal growth of the seedling root, but the RNAi lines of OsNAC45 were more sensitive. The roots of the OE lines accumulated less superoxide anion under low temperature stress and more hydrogen peroxide under normal conditions than WT. After NaCl treatment, the Peroxidase (POD) assay showed POD activity of the roots in OE lines were elevated, but were decreased in RNAi lines and WT; additionally, the increment of lignin content in OE lines was significantly higher than RNAi lines and WT in roots. Furthermore, expression levels of some rice root development-related genes were altered in the transgenic plants. Under cold and salt stresses, expression of NCED4, a key enzyme in abscisic acid synthesis, was decreased in OE lines and raised in RNAi lines compared to WT. The data suggests that OsNAC45 may participate in the response of cold/salt stresses by lessening the accumulation of reactive oxygen species and increasing lignin in rice roots thus alleviating the restraint to the growth of seedling root. Overexpression of OsNAC45 weakened the high temperature tolerance; however, inhibiting its expression could improve the tolerance. Together, these results indicate that OsNAC45 plays an important role in the coordination, development, and response of rice plants under abiotic stresses.

Increasing agricultural productivity is one of the most important goals of plant science research and imperative to meet the needs of a rapidly growing population. Rice (Oryza sativa L.) is one of the most important staple crops worldwide. Grain size is both a major determinant of grain yield in rice and a target trait for domestication and artificial breeding. Here, a genome-wide association study of grain length and grain width was performed using 996,722 SNP markers in 270 rice accessions. Five and four quantitative trait loci were identified for grain length and grain width, respectively. In particular, the novel grain size gene OsSNB was identified from qGW7, and further results showed that OsSNB negatively regulated grain size. Most notably, knockout mutant plants by CRISPR/Cas9 technology showed increased grain length, width, and weight, while overexpression of OsSNB yielded the opposite. Sequencing of this gene from the promoter to the 3’-untranslated region in 168 rice accessions from a wide geographic range identified eight haplotypes. Furthermore, Hap 3 has the highest grain width discovered in japonica subspecies. Compared to other haplotypes, Hap 3 has a 225 bp insertion in the promoter. Based on the difference between Hap 3 and other haplotypes, OsSNB_Indel2 was designed as a functional marker for the improvement of rice grain width. This could be directly used to assist selection toward an improvement of grain width. These findings suggest OsSNB as useful for further improvements in yield characteristics in most cultivars., Author summary Grain weight, including grain length and grain width, is a complex trait, and hundreds of quantitative trait loci (QTLs) were detected in different genetic rice populations. However, only about 10 genes have been isolated and characterized until now. Nine QTLs for grain size were identified by genome-wide association study in a natural rice population. The novel grain size gene OsSNB was identified from qGW7 based on the difference of expression levels between two different varieties with significantly different grain width. OsSNB is an AP2 transcription factor that is negatively regulated grain size. However, OsSNB was found to regulate the transition from the spikelet meristem to the floral meristem and the floral organ development in previous study. Compared to other haplotypes, Hap 3 has a 225 bp insertion in the promoter. Based on the difference between Hap 3 and other haplotypes, OsSNB_Indel2 was designed as a functional marker for the improvement of rice grain width. This can be directly used to assist selection for grain width improvement.

We identified a new dehydration-responsive element-binding protein gene, OsDREB6, which is an A-6 subgroup member of the DREB subfamily, of AP2/EREBP proteins, in Oryza sativa ssp. Japonica cv. Nipponbare. Expression pattern analysis indicated that OsDREB6 was induced in response to dehydration, high salinity, and most importantly by cold stress (4°C). Analysis using the yeast one-hybrid system showed that OsDREB6 had transcriptional activation activity and bound specifically to the DRE ciselement. Overexpression of OsDREB6 increased tolerance to osmotic, high salt, and cold stresses in transgenic rice whereas, OsDREB6 double-stranded RNA interference (RNAi) lines were found to be more sensitive to these stresses than wild-type rice. Additionally, sugar, proline and catalase levels in OsDREB6 RNAi lines were significantly lower than in the corresponding OsDREB6 overexpression lines and control lines after PEG treatment. In contrast, malondialdehyde (MDA) levels were significantly higher in RNAi lines than in control and overexpression lines. Furthermore, expression levels of some stress-related genes were altered in the transgenic plants. The data suggests that OsDREB6 may participate in the stress response by mediating the expression of specific genes, and may also be involved plant metabolism. Together, these results indicated that OsDREB6 plays an important role in the response of plants to a wide range of osmotic stresses.

The mitogen-activated protein kinase kinases (MKK) represent a small gene family that is located at the center of the MAPK cascade and play an important role in responses to biotic and abiotic stresses and in plant growth and development. Here, we report the cloning of an MKK gene from Brassica napus, BnMKK1 (GenBank Accession No. HQ916282), by RT-PCR. The BnMKK1 cDNA is 1,447 bp in length with an open reading frame of 1,092 bp. The gene encodes a putative MKK protein that contains a conserved motif S/TxxxxxS/T (where x represents any amino acid) and a MPK docking domain in its N-terminal extension. The orthologues of the BnMKK1 protein are highly conserved among mosses, ferns, dicotyledons and monocotyledons. Southern hybridization revealed the presence of more than two copies of the BnMKK1 homologues in the genome of B. napus. Quantitative reverse transcription–polymerase chain reaction analyses showed that the BnMKK1 transcripts accumulated in response to cold, ABA (abscisic acid) and MeJA (methyl jasmonate) but declined in response to mannitol, NaCl, H2O2, and SA (salicylic acid). The inhibitors of MAPK activation, PD98059 and U0126, did not inhibit BnMKK1 transcription. BnMKK1 transgenic tobacco plants grew slower and showed significantly delayed flower times compared to the wild type. Their root development was insensitive to treatment with 100 mM IAA (indole-3-acetic acid). The detached leaves from the transgenic BnMKK1 tobacco plants strengthened the inhibition to bacterial development at later growth stages. The overexpression of BnMKK1 leads to rapid water loss and enhanced sensitivities to drought stress in transgenic tobacco plants. These results show that BnMKK1 plays an important role in the response of plants to pathogenic bacteria and drought stress.

In eukaryotes, small noncoding RNA molecules of 16–29 nucleotides in length play crucial roles in the regulation of gene expression. Some 377 sequences representing rice pseudo-microRNAs (miRNAs) are available in release 13.0 of the miRBase sequence database ( http://microrna.sanger.ac.uk/sequences/index.shtml ) and are grouped into 143 families. Most newly deposited miRNA sequences are likely to be species-specific. To understand the relationship between miRNAs and transposable elements (TEs) in rice, the RepeatMasker application ( http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker ) was used to screen single-stranded precursor miRNA (pre-miRNA) sequences. This analysis revealed that 33.1% of miRNAs and 36.4% of miRNA families are associated with interspersed repeats, and most of them are species-specific. Furthermore, multiple miRNA families can be encoded by the same TE class. Alignment analysis revealed that miR439 originated from an MuDR4-OS TE, which amplified and diversified in the genome as an inverted repeat of the core sequence followed by multiple repeats. Multiple copies of miR445 and its complexity originate from and are driven by the DNA/Tourist TE class. These results provide an important contribution to the elucidation of TE-driven mechanisms that regulate the species specificity and complexity of rice miRNAs.

This study describes the development of electrofusion techniques using the ‘donor-recipient’ model for the production of cybrids between common cultivated winter wheat (Triticum aestivum L.) cv. Jinghua No. 1 and a phylogenetically remote, sexually incompatible grass species, Italian ryegrass (Lolium multiflorum Lam.), which belong to two different subtribes: Triticinae and Loliinae. Wheat protoplasts were metabolically inactivated by iodoacetamide before fusion, while protoplasts of Italian ryegrass were X-ray irradiated before protoplast isolation. The suspension cells were directly used to optizmize the inactivation parameters. By exploring the minimum irreversible membrane breakdown strength, the electrofusion parameters were optimized just a few minutes before electrofusion began. A total of 108 green plantlets were obtained, and about half of the green plants uncontrollably necrotized. Among all green plants, 14 were rooted normally and transplanted in growth chamber or field and developed to maturity. All these transplanted plants were male sterile with smaller and off-white anthers. Seeds were obtained by crossing with Jinghua No. 1. Three transplanted regenerants possessed the characteristics of glume facing the rachis, which was the taxonomic characteristic distinguishing the two subtribes of Triticinae and Lolliinae. Although Southern blot hybridization analysis of 33 randomly selected regenerants using a wheat ribosomal DNA probe (pHA71) did not find any differences to wheat, analysis using two mitochondrial probes B342 (cox l), 490 (Pro II) and one chloroplastidic probe pHve H5 revealed that 31 plants were ‘true cybrids’ by showing ryegrass-specific band(s) or new band(s). It also showed that the mitochondria and chloroplasts were not coexistent as the restriction fragment length polymorphism band of Italian ryegrass was not detected by the mitochondrial probes 7 (26s), B342 (cox I), pHJ2-7-1 (cox II), B30 (atp9), and the chloroplast probe pHvc P5. To regenerate the cybrids, the regeneration capacity of the recipient (wheat) was crucial in this study.

A gene encoding mitogen-activated protein kinase was isolated from oilseed rape (Brassica napus) following drought treatment. The cloned gene, designated as BnMPK3 (GenBank accession no. AY642433 ), contains one phosphorylation motif (TEY) and a conserved CD domain in its C-terminal extension. BnMPK3 is highly homologous to AtMPK3 gene from Arabidopsis thaliana and belongs to the A-subgroup of mitogen-activated protein kinase. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that the expression level of BnMPK3 transcript was up-regulated by mannitol, NaCl, cold, H2O2, abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate acid (m-JA). Interestingly, SA, m-JA enhanced gradually BnMPK3's expression all along after 20–60 min treatment, but its expression was all initiated by mannitol, NaCl, H2O2 and ABA in a short time and reached the highest level after 1–2 h treatment. Hysteresis of cold response showed that BnMPK3's expression was initiated after 1 h from normal growth conditions at room temperature to 4 °C, and reached the highest level after 6 h treatment. The inhibitors of MAPK activation PD98059 and U0126 did not inhibit BnMPK3 transcript. BnMPK3 was cloned into pYES2.1 and transformed into yeasts, and BnMPK3's expression enhanced yeasts’ resistance to mannitol and peroxide in 600 mM mannitol and 0.2 mM tBuOOH drug assays. The present studies show that BnMPK3 is an early response gene involved in responses to biotic and abiotic stresses.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS , GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H 2 O 2 ). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea .

A new stress-responsive gene BoRS1 (GenBank accession No. AY373021) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with LTI65, RD29A, RD29B and COR78 from Arabidopsis thaliana. Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea. Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.

The drought tolerance (DT) of plants is a complex quantitative trait. Under natural and artificial selection, drought tolerance represents the crop survival ability and production capacity under drought conditions (Luo, 2010). To understand the regulation mechanism of varied drought tolerance among rice genotypes, 95 diverse rice landraces or varieties were evaluated within a field screen facility based on the ‘line–source soil moisture gradient’, and their resistance varied from extremely resistant to sensitive. The method of Ecotype Targeting Induced Local Lesions in Genomes (Ecotilling) was used to analyze the diversity in the promoters of 24 transcription factor families. The bands separated by electrophoresis using Ecotilling were converted into molecular markers. STRUCTURE analysis revealed a value of K = 2, namely, the population with two subgroups (i.e., indica and japonica), which coincided very well with the UPGMA clusters (NTSYS-pc software) using distance-based analysis and InDel markers. Then the association analysis between the promoter diversity of these transcription factors and the DT index/level of each variety was performed. The results showed that three genes were associated with the DT index and that five genes were associated with the DT level. The sequences of these associated genes are complex and variable, especially at approximately 1000 bp upstream of the transcription initiation sites. The study illuminated that association analysis aimed at Ecotilling diversity of natural groups could facilitate the isolation of rice genes related to complex quantitative traits.