Nitrogen inhibition of nitrogenase activity involves the modulation of cytosolic invertase in soybean nodule.

Legume symbiotic nitrogen fixation (SNF) is suppressed by inorganic N in the soil. High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules. However, the underlying molecular mechanisms remain unclear. Here, we identify GmCIN1 which encodes a cytosolic invertase, as a gateway for the N-tuning of sucrose utilization in nodules. GmCIN1 is enriched in mature soybean nodules and its expression is regulated by nitrogen status. The knockout of GmCIN1 using genome editing partially mimicks the inhibitory effects of N on nitrogenase activity and sugar content and the impact of high N on nodule transcriptomes. This indicates that GmCIN1 partially mediates the high N inhibition of nodule activity. Moreover, ChIP-qPCR and EMSA reveal that SNAP1/2 transcription factors directly bind to the GmCIN1 promoter. In addition, SNAP1/2 may be involved in the repression of GmCIN1 expression in mature nodules at high N concentrations. Our findings provide insights into the involvement of the transcriptional tuning of C metabolism genes by N-signaling modulators in the N-induced inhibition of nitrogenase activity.

Genetically optimizing soybean nodulation improves yield and protein content.

Symbiotic nitrogen fixation in legume nodules requires substantial energy investment from host plants, and soybean (Glycine max (L.) supernodulation mutants show stunting and yield penalties due to overconsumption of carbon sources. We obtained soybean mutants differing in their nodulation ability, among which rhizobially induced cle1a/2a (ric1a/2a) has a moderate increase in nodule number, balanced carbon allocation, and enhanced carbon and nitrogen acquisition. In multi-year and multi-site field trials in China, two ric1a/2a lines had improved grain yield, protein content and sustained oil content, demonstrating that gene editing towards optimal nodulation improves soybean yield and quality.

The NAC transcription factors SNAP1/2/3/4 are central regulators mediating high nitrogen responses in mature nodules of soybean.

Legumes can utilize atmospheric nitrogen via symbiotic nitrogen fixation, but this process is inhibited by high soil inorganic nitrogen. So far, how high nitrogen inhibits N2 fixation in mature nodules is still poorly understood. Here we construct a co-expression network in soybean nodule and find that a dynamic and reversible transcriptional network underlies the high N inhibition of N2 fixation. Intriguingly, several NAC transcription factors (TFs), designated as Soybean Nitrogen Associated NAPs (SNAPs), are amongst the most connected hub TFs. The nodules of snap1/2/3/4 quadruple mutants show less sensitivity to the high nitrogen inhibition of nitrogenase activity and acceleration of senescence. Integrative analysis shows that these SNAP TFs largely influence the high nitrogen transcriptional response through direct regulation of a subnetwork of senescence-associated genes and transcriptional regulators. We propose that the SNAP-mediated transcriptional network may trigger nodule senescence in response to high nitrogen.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9.

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T2 progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean.

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR-Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR-Cas9 as a mutant screening tool. Here, we report a pooled CRISPR-Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR-Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.

Transcriptome analysis by Illumina high-throughout paired-end sequencing reveals the complexity of differential gene expression during in vitro plantlet growth and flowering in Amaranthus tricolor L.

Amaranthus tricolor L. is a C4 plant, which is consumed as a major leafy vegetable in some tropical countries. Under conditions of high temperature and short daylight, Am. tricolor readily bolts and blooms, degrading leaf quality. A preliminary in vitro flowering study demonstrated that the flowering control pathway in Am. tricolor may differ from that of Arabidopsis. Nevertheless, no transcriptome analysis of the flowering process in Amaranthus has been conducted. To study Am. tricolor floral regulatory mechanisms, we conducted a large-scale transcriptome analysis--based on Illumina HiSeq sequencing of cDNA libraries generated from Am. tricolor at young seedling (YSS), adult seedling (ASS), flower bud (FBS), and flowering (FS) stages. A total of 99,312 unigenes were obtained. Using BLASTX, 43,088 unigenes (43.39%) were found to have significant similarity with accessions in Nr, Nt, and Swiss-Prot databases. Of these unigenes, 11,291 were mapped to 266 KEGG pathways. Further analysis of the four digital transcriptomes revealed that 735, 17,184, 274, and 206 unigenes were specifically expressed during YSS, ASS, FBS, and FS, respectively, with 59,517 unigenes expressed throughout the four stages. These unigenes were involved in many metabolic pathways related to in vitro flowering. Among these pathways, 259 unigenes were associated with ubiquitin-mediated proteolysis, indicating its importance for in vitro flowering in Am. tricolor. Other pathways, such as circadian rhythm and cell cycle, also had important roles. Finally, 26 unigenes were validated by qRT-PCR in samples from Am. tricolor at YSS, ASS, FBS, and FS; their differential expressions at the various stages indicate their possible roles in Am. tricolor growth and development, but the results were somewhat similar to Arabidopsis. Because unigenes involved in many metabolic pathways or of unknown function were revealed to regulate in vitro plantlet growth and flowering in Am. tricolor, the process appears to be highly complex in this species.