Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. In this study, we identified and characterized a gene, GmNFYA3, which encodes the NF-YA subunit of the NF-Y complex in soybeans (Glycine max L.). Real time RT-PCR analysis indicated that GmNFYA3 was induced by abscisic acid (ABA) and abiotic stresses, such as polyethylene glycol, NaCl and cold. Subcellular localization analysis suggested that GmNFYA3 may activate its specific targets in the nucleus. Histochemical β-glucuronidase (GUS) staining revealed that the expression of the GUS gene driven by the GmNFYA3 promoter occurred in various transgenic Arabidopsis tissues. Coexpression in Nicotiana benthamiana and 5' RACE assays indicated that miR169 directs GmNFYA3 mRNA cleavage in vivo. Overexpression of GmNFYA3 resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance. In addition, the transgenic Arabidopsis exhibited increased sensitivity to high salinity and exogenous ABA. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI1, ABI2) and stress-responsive genes, including RD29A and CBF3, were generally higher in GmNFYA3 plants than in wild-type controls under normal conditions. These results suggest that the GmNFYA3 gene functions in positive modulation of drought stress tolerance and has potential applications in molecular breeding to enhance drought tolerance in crops.

Spike number per unit area, number of grains per spike, and thousand kernel weight (TKW) are important yield components. In China, increases in wheat (Triticum aestivum) yields are mainly due to increases in grain number per spike and TKW. TKW mainly depends on starch content, as starch accounts for about 70% of the grain endosperm. Sucrose synthase catalysis is the first step in the conversion of sucrose to starch, that is, the conversion of sucrose to fructose and UDP-glucose by the wheat sucrose synthase genes (TaSus1 and TaSus2) that are located on chromosomes 7A/7B/7D and 2A/2B/2D, respectively. A total of 1,520 wheat accessions were genotyped at the six loci. Two, two, five, and two haplotypes were identified at the TaSus2-2A, TaSus2-2B, TaSus1-7A, and TaSus1-7B loci, respectively. Their main variations were detected within the introns. Significant differences between the haplotypes correlated with TKW differences among 348 modern Chinese cultivars from the core collection. Frequency changes for favored haplotypes showed gradual increases in cultivars released since beginning of the last century in China, Europe, and North America. Geographic distributions and time changes of favored haplotypes were characterized in six major wheat production regions worldwide. Strong selection bottlenecks to haplotype variations occurred at polyploidization and domestication and during breeding of wheat. Genetic-effect differences between haplotypes at the same locus influence the selection time and intensity. This work shows that the endosperm starch synthesis pathway is a major target of indirect selection in global wheat breeding for higher yield.

Sucrose synthase catalyzes the reaction sucrose + UDP → UDP-glucose + fructose, the first step in the conversion of sucrose to starch in endosperm. Previous studies identified two tissue-specific, yet functionally redundant, sucrose synthase (SUS) genes, Sus1 and Sus2. In the present study, the wheat Sus2 orthologous gene (TaSus2) series was isolated and mapped on chromosomes 2A, 2B, and 2D. Based on sequencing in 61 wheat accessions, three single-nucleotide polymorphisms (SNPs) were detected in TaSus2-2B. These formed two haplotypes (Hap-H and Hap-L), but no diversity was found in either TaSus2-2A or TaSus2-2D. Based on the sequences of the two haplotypes, we developed a co-dominant marker, TaSus2-2B ( tgw ), which amplified 423 or 381-bp fragments in different wheat accessions. TaSus2-2B ( tgw ) was located between markers Xbarc102.2 and Xbarc91 on chromosome 2BS in a RIL population from Xiaoyan 54 × Jing 411. Association analysis suggested that the two haplotypes were significantly associated with 1,000 grain weight (TGW) in 89 modern wheat varieties in the Chinese mini-core collection. Mean TGW difference between the two haplotypes over three cropping seasons was 4.26 g (varying from 3.71 to 4.94 g). Comparative genomics analysis detected major kernel weight QTLs not only in the chromosome region containing TaSus2-2B (tgw), but also in the collinear regions of TaSus2 on rice chromosome 7 and maize chromosome 9. The preferred Hap-H haplotype for high TGW underwent very strong positive selection in Chinese wheat breeding, but not in Europe. The geographic distribution of Hap-H was perhaps determined by both latitude and the intensity of selection in wheat breeding.

RNA silencing such as quelling and meiotic silencing by unpaired DNA (MSUD) and several other classes of special small RNAs have been discovered in filamentous fungi recently. More than four different mechanisms of microRNA-like RNAs (milRNAs) production have been illustrated in the model fungus Neurospora crassa including a dicer-independent pathway. To date, very little work focusing on small RNAs in fungi has been reported and no universal or particular characteristic of milRNAs were defined clearly. In this study, small RNA and degradome libraries were constructed and subsequently deep sequenced for investigating milRNAs and their potential cleavage targets on the genome level in the filamentous fungus F. oxysporum f. sp. lycopersici. As a result, there is no intersection of conserved miRNAs found by BLASTing against the miRBase. Further analysis showed that the small RNA population of F. oxysporum shared many common features with the small RNAs from N. crassa and other fungi. According to the known standards of miRNA prediction in plants and animals, milRNA candidates from 8 families (comprising 19 members) were screened out and identified. However, none of them could trigger target cleavage based on the degradome data. Moreover, most major signals of cleavage in transcripts could not match appropriate complementary small RNAs, suggesting that other predominant modes for milRNA-mediated gene regulation could exist in F. oxysporum. In addition, the PAREsnip program was utilized for comprehensive analysis and 3 families of small RNAs leading to transcript cleavage were experimentally validated. Altogether, our findings provided valuable information and important hints for better understanding the functions of the small RNAs and milRNAs in the fungal kingdom.

Wheat anther-specific invertase genes were haplotyped in wheat. Strong allelic selection occurred during wheat polyploidization, domestication and breeding because of their association with yield traits. Plant invertase hydrolyzes sucrose into glucose and fructose. Cell wall invertase (CWI), one of the three types of invertase, is essential for plant development. Based on isolated TaCWI genes from chromosomes 4A, 5B and 5D, two SNPs were detected in the promoter region of TaCWI-4A, and four SNPs and two Indels were present in the TaCWI-5D gene. No polymorphism was detected in TaCWI-5B coding or promoter regions. CAPS markers caps4A and caps5D were developed to discriminate haplotypes of TaCWI-4A and TaCWI-5D. Marker/trait association analysis indicated that Hap-5D-C at TaCWI-5D was significantly associated with higher thousand kernel weight (TKW) in 348 Chinese modern cultivars grown in multiple environments. Geographic distributions and changes over time of favored haplotypes showed that Hap-5D-C was the most frequent haplotype in modern cultivars and was strongly positively selected in six major wheat production regions worldwide. However, selection for haplotypes at TaCWI-4A was not so evident, possibly due to balancing effects of the two haplotypes on TKW and grain number per spike (GN). In rainfed production regions, Hap-4A-C was favored because it brought more seeds, but in well irrigated conditions, Hap-4A-T was favored in modern breeding because of higher TKW. Evolutionary analysis among wheat and its relatives showed that genetic diversity of TaCWI genes on chromosomes 4A and 5D declined dramatically in progression from the diploid level to modern polyploid cultivars. There was strong allelic selection during polyploidization, domestication and breeding.

Key message We found GmNAC06 plays an important role in salt stress responses through the phenotypic, physiological and molecular analyses of OE, VC, and Mutant composite soybean. Abstract Salinization affects 20% of all cultivated land worldwide because of the high salinity of irrigation water and the excessive use of water, and this amount is increasing daily. NAC (NAM, ATAF, and CUC) have been found to be involved in salt stress. In this study, a soybean NAC gene, GmNAC06 (Glyma06g21020.1), was cloned and functionally characterized. The results of expression analysis suggested that salt stress could influence the expression level of GmNAC06. The subcellular localization analysis results suggested that GmNAC06 may function as a transcription factor. Under salt stress, the overexpression technology combined with CRISPR-Cas9 system found that GmNAC06 could cause the accumulation of proline and glycine betaine to alleviate or avoid the negative effects of ROS; similarly, it could control the Na+/K+ ratios in hairy roots to maintain ionic homeostasis. The fresh weight of the transgenic hairy roots and the histochemical ROS staining of wild leaves suggested that transgenic hairy roots influence the function of wild leaves under salt stress conditions. Moreover, the expression levels of GmUBC2 and GmHKT1 were higher in the GmNAC06 hairy roots than in the control. Thus, the overexpression of GmNAC06 in hairy roots notably causes an entire composite plant to exhibit salt tolerance. The phenotype of composite soybean plants and transgenic Arabidopsis plants suggest that GmNAC06 plays a role in response to salt stress and could be useful in generating salt tolerant transgenic crops.

Background Environmental stimuli can activate a series of physiological and biochemical responses in plants accompanied by extensive transcriptional reprogramming. Long non-coding RNAs (lncRNAs), as versatile regulators, control gene expression in multiple ways and participate in the adaptation to biotic and abiotic stresses. Results In this study, soybean seedlings were continuously cultured for 15 days with high salinity solutions started from seed germination. Strand-specific whole transcriptome sequencing and stringent bioinformatic analysis led to the identification of 3030 long intergenic non-coding RNAs (lincRNAs) and 275 natural antisense transcripts (lncNATs) in soybean roots. In contrast to mRNAs, newly identified lncRNAs exhibited less exons, similar AU content to UTRs, even distribution across the genome and low evolutionary conservation. Remarkably, more than 75% of discovered lncRNAs that were activated or up-regulated by continuous salt stress mainly targeted proteins with binding and catalytic activities. Furthermore, two DNA methylation maps with single-base resolution were generated by using reduced representation bisulfite sequencing, offering a genome-wide perspective and important clues for epigenetic regulation of stress-associated lncRNAs and protein-coding genes. Conclusions Taken together, our findings systematically demonstrated the characteristics of continuous salt stress-induced lncRNAs and extended the knowledge of corresponding methylation profiling, providing valuable evidence for a better understanding of how plants cope with long-term salt stress circumstances.

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