Shoot-to-root translocated GmNN1/FT2a triggers nodulation and regulates soybean nitrogen nutrition.

Symbiotic nitrogen fixation (SNF) provides sufficient nitrogen (N) to meet most legume nutrition demands. In return, host plants feed symbionts carbohydrates produced in shoots. However, the molecular dialogue between shoots and symbionts remains largely mysterious. Here, we report the map-based cloning and characterization of a natural variation in GmNN1, the ortholog of Arabidopsis thaliana FLOWERING LOCUS T (FT2a) that simultaneously triggers nodulation in soybean and modulates leaf N nutrition. A 43-bp insertion in the promoter region of GmNN1/FT2a significantly decreased its transcription level and yielded N deficiency phenotypes. Manipulating GmNN1/GmFT2a significantly enhanced soybean nodulation, plant growth, and N nutrition. The near-isogenic lines (NILs) carrying low mRNA abundance alleles of GmNN1/FT2a, along with stable transgenic soybeans with CRISPR/Cas9 knockouts of GmNN1/FT2a, had yellower leaves, lower N concentrations, and fewer nodules than wild-type control plants. Grafting together with split-root experiments demonstrated that only shoot GmNN1/FT2a was responsible for regulating nodulation and thereby N nutrition through shoot-to-root translocation, and this process depends on rhizobial infection. After translocating into roots, shoot-derived GmNN1/FT2a was found to interact with GmNFYA-C (nuclear factor-Y subunit A-C) to activate symbiotic signaling through the previously reported GmNFYA-C-ENOD40 module. In short, the description of the critical soybean nodulation regulatory pathway outlined herein sheds novel insights into the shoot-to-root signaling required for communications between host plants and root nodulating symbionts.

Cooperative interactions between nitrogen fixation and phosphorus nutrition in legumes.

Legumes such as soybean are considered important crops as they provide proteins and oils for humans and livestock around the world. Different from other crops, leguminous crops accumulate nitrogen (N) for plant growth through symbiotic nitrogen fixation (SNF) in coordination with rhizobia. A number of studies have shown that efficient SNF requires the cooperation of other nutrients, especially phosphorus (P), a nutrient deficient in most soils. During the last decades, great progress has been made in understanding the molecular mechanisms underlying the interactions between SNF and P nutrition, specifically through the identification of transporters involved in P transport to nodules and bacteroids, signal transduction, and regulation of P homeostasis in nodules. These studies revealed a distinct N-P interaction in leguminous crops, which is characterized by specific signaling cross talk between P and SNF. This review aimed to present an updated picture of the cross talk between N fixation and P nutrition in legumes, focusing on soybean as a model crop, and Medicago truncatula and Lotus japonicus as model plants. We also discuss the possibilities for enhancing SNF through improving P nutrition, which are important for high and sustainable production of leguminous crops.

The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis.

BACKGROUND: WRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata. RESULTS: In this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five ß-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants. CONCLUSION: These results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

Overexpression of GmPHR1 Promotes Soybean Yield through Global Regulation of Nutrient Acquisition and Root Development.

MYB-CC transcription factors (TFs) are essential for plant growth and development. Members of the MYB-CC subfamily with long N terminal domains, such as phosphate starvation response 1 (PHR1) or PHR1-like TFs, have well documented functions, while those with short N terminal domains remain less understood. In this study, we identified a nodule specific MYB-CC transcription factor 1 (GmPHR1) in soybean that is different from other canonical PHR family genes in that GmPHR1 harbors a short N terminal ahead of its MYB-CC domain and was highly induced by rhizobium infection. The overexpression of GmPHR1 dramatically increased the ratio of deformed root hairs, enhanced subsequent soybean nodulation, and promoted soybean growth in pot experiments. The growth promotion effects of GmPHR1 overexpression were further demonstrated in field trails in which two GmPHR1-OE lines yielded 10.78% and 8.19% more than the wild type line. Transcriptome analysis suggested that GmPHR1 overexpression led to global reprogramming, with 749 genes upregulated and 279 genes downregulated, especially for genes involved in MYB transcription factor activities, root growth, and nutrient acquisition. Taken together, we conclude that GmPHR1 is a key gene involved in the global regulation of nodulation, root growth, and nutrient acquisition in soybeans, and is thus a promising candidate gene to target for soybean yield enhancement.

Cloning and characterization of KoOsmotin from mangrove plant Kandelia obovata under cold stress.

BACKGROUND: Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution. Kandelia obovata is the most cold-resistance specie in mangrove plants, but little is known about the molecular mechanism underlying its resistance to cold. Osmotin is a key protein associated with abiotic and biotic stress response in plants but no information about this gene in K. obovata was reported. RESULTS: In this study, a cDNA sequence encoding osmotin, KoOsmotin (GenBank accession no. KP267758), was cloned from mangrove plant K. obovata. The KoOsmotin protein was composed of 221 amino acids and showed a calculated molecular mass of 24.11 kDa with pI 4.92. The KoOsmotin contained sixteen cysteine residues and an N-terminal signal peptide, which were common signatures to most osmotins and pathogenesis-related 5 proteins. The three-dimensional (3D) model of KoOsmotin, contained one α-helix and eleven ß-strands, was formed by three characteristic domains. Database comparisons of the KoOsmotin showed the closest identity (55.75%) with the osmotin 34 from Theobroma cacao. The phylogenetic tree also revealed that the KoOsmotin was clustered in the branch of osmotin/OLP (osmotin-like protien). The KoOsmotin protein was proved to be localized to both the plasma membrane and cytoplasm by the subcellular localization analysis. Gene expression showed that the KoOsmotin was induced primarily and highly in the leaves of K. obovata, but less abundantly in stems and roots. The overexpressing of KoOsmotin conferred cold tolerance in Escherichia coli cells. CONCLUSION: As we known, this is the first study to explore the osmotin of K. obovata. Our study provided valuable clues for further exploring the function of KoOsmotin response to stress.

miR169c-NFYA-C-ENOD40 modulates nitrogen inhibitory effects in soybean nodulation.

Legume crops contribute a great portion of clean nitrogen (N) to agro-ecosystems through symbiotic N2 fixation in the nodule; however, the nodulation is always inhibited by high N availability which is known as the N inhibitory effect through largely unknown mechanisms. We functionally investigated miR169c-GmNFYA-C-GmENOD40 under multiple N conditions in soybean (Glycine max) (ENOD, Early Nodulin; NFYA, Nuclear Factor-Y Subunit A). We elucidated their regulatory roles in soybean nodulation through analyzing expression patterns, micro-messenger RNA (miRNA-mRNA) interactions, phenotypes of transgenic soybean plants and genetic interactions. We found that miR169c expression was induced by high N, whereas its target GmNFYA-C was preferentially expressed in nodules and induced by rhizobium inoculation. Overexpression of miR169c inhibited nodulation through targeting 3'-UTR of GmNFYA-C, whereas knockout miR169c through CRISPR-cas9 promoted nodulation. However, overexpression of GmNFYA-C promoted soybean nodulation through facilitating rhizobium infection and increasing the expression of symbiotic signaling gene GmENOD40. Besides, GmNFYA-C directly induced the expression of GmENOD40. In addition, overexpression of GmNFYA-C without the target site of miR169c partially attenuated the inhibitory effect of high N on soybean nodulation. We discovered a new regulatory pathway involving the miR169c-NFYA-C-ENOD40 module that regulates soybean nodulation in response to N availability. This pathway provides substantial new insights into the mechanisms underlying the N inhibitory effect on nodulation.

Quantitative Proteomics Reveals that GmENO2 Proteins Are Involved in Response to Phosphate Starvation in the Leaves of Glycine max L.

Soybean (Glycine max L.) is a major crop providing important source for protein and oil for human life. Low phosphate (LP) availability is a critical limiting factor affecting soybean production. Soybean plants develop a series of strategies to adapt to phosphate (Pi) limitation condition. However, the underlying molecular mechanisms responsible for LP stress response remain largely unknown. Here, we performed a label-free quantification (LFQ) analysis of soybean leaves grown under low and high phosphate conditions. We identified 267 induced and 440 reduced differential proteins from phosphate-starved leaves. Almost a quarter of the LP decreased proteins are involved in translation processes, while the LP increased proteins are accumulated in chlorophyll biosynthetic and carbon metabolic processes. Among these induced proteins, an enolase protein, GmENO2a was found to be mostly induced protein. On the transcriptional level, GmENO2a and GmENO2b, but not GmENO2c or GmENO2d, were dramatically induced by phosphate starvation. Among 14 enolase genes, only GmENO2a and GmENO2b genes contain the P1BS motif in their promoter regions. Furthermore, GmENO2b was specifically induced in the GmPHR31 overexpressing soybean plants. Our findings provide molecular insights into how soybean plants tune basic carbon metabolic pathway to adapt to Pi deprivation through the ENO2 enzymes.

Functional assembly of root-associated microbial consortia improves nutrient efficiency and yield in soybean.

Root-associated microbes are critical for plant growth and nutrient acquisition. However, scant information exists on optimizing communities of beneficial root-associated microbes or the mechanisms underlying their interactions with host plants. In this report, we demonstrate that root-associated microbes are critical influencers of host plant growth and nutrient acquisition. Three synthetic communities (SynComs) were constructed based on functional screening of 1,893 microbial strains isolated from root-associated compartments of soybean plants. Functional assemblage of SynComs promoted significant plant growth and nutrient acquisition under both N/P nutrient deficiency and sufficiency conditions. Field trials further revealed that application of SynComs stably and significantly promoted plant growth, facilitated N and P acquisition, and subsequently increased soybean yield. Among the tested communities, SynCom1 exhibited the greatest promotion effect, with yield increases of up to 36.1% observed in two field sites. Further RNA-seq implied that SynCom application systemically regulates N and P signaling networks at the transcriptional level, which leads to increased representation of important growth pathways, especially those related to auxin responses. Overall, this study details a promising strategy for constructing SynComs based on functional screening, which are capable of enhancing nutrient acquisition and crop yield through the activities of beneficial root-associated microbes.

Genotype and rhizobium inoculation modulate the assembly of soybean rhizobacterial communities.

Rhizosphere bacterial communities are vital for plants, yet the composition of rhizobacterial communities and the complex interactions between roots and microbiota, or between microbiota, are largely unknown. In this study, we investigated the structure and composition of rhizobacterial communities in two soybean cultivars and their recombinant inbred lines contrasting in nodulation through 16S rRNA amplicon sequencing in two years of field trials. Our results demonstrate that soybean plants are able to select microbes from bulk soils at the taxonomic and functional level. Soybean genotype significantly influenced the structure of rhizobacterial communities and resulted in dramatically different co-occurrence networks of rhizobacterial communities between different genotypes of soybean plants. Furthermore, the introduction of exogenous rhizobia through inoculation altered soybean rhizobacterial communities in genotype-dependent manner. Rhizobium inoculation not only stimulated the proliferation of potential beneficial microbes but also increased connections in rhizobacterial networks and changed the hub microbes, all of which led to the association of distinctive bacterial communities. Taken together, we demonstrated that the assembly of soybean rhizobacterial communities was determined by both genotype and the introduction of exogenous rhizobia. These findings bolster the feasibility of root microbiome engineering through inoculation of specific microbial constituents.

Rice SPX6 negatively regulates the phosphate starvation response through suppression of the transcription factor PHR2.

Phosphorus (P) is an essential macronutrient for plant growth and development, but the molecular mechanism determining how plants sense external inorganic phosphate (Pi) levels and reprogram transcriptional and adaptive responses is incompletely understood. In this study, we investigated the function of OsSPX6 (hereafter SPX6), an uncharacterized member of SPX domain (SYG1, Pho81 and XPR1)-containing proteins in rice, using reverse genetics and biochemical approaches. Transgenic plants overexpressing SPX6 exhibited decreased Pi concentrations and suppression of phosphate starvation-induced (PSI) genes. By contrast, transgenic lines with decreased SPX6 transcript levels or spx6 mutant showed significant Pi accumulation in the leaf and upregulation of PSI genes. Overexpression of SPX6 genetically suppressed the overexpression of PHOSPHATE STARVATION RESPONSE REGULATOR 2 (PHR2) in terms of the accumulation of high Pi content. Moreover, direct interaction of SPX6 with PHR2 impeded PHR2 translocation into the nucleus, and inhibited PHR2 binding to the P1BS (PHR1 binding sequence) element. SPX6 protein was degraded in leaves under Pi-deficient conditions, whereas it accumulated in roots. We conclude that rice SPX6 is another important negative regulator in Pi starvation signaling through the interaction with PHR2. SPX6 shows different responses to Pi starvation in shoot and root, which differ from those of other SPX proteins.

SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through Its Interaction with PHR2 in Rice.

PHR2, a central regulator of phosphate signaling in rice, enhanced the expression of phosphate starvation-induced (PSI) genes and resulted in the enhancement of Pi acquisition under Pi deficiency stress. This occurred via PHR2 binding to a cis-element named the PHR1 binding sequences. However, the transcription level of PHR2 was not responsive to Pi starvation. So how is activity of transcription factor PHR2 adjusted to adapt diverse Pi status? Here, we identify an SPX family protein, Os-SPX4 (SPX4 hereafter), involving in Pi starvation signaling and acting as a negative regulator of PHR2. SPX4 is shown to be a fast turnover protein. When Pi is sufficient, through its interaction with PHR2, SPX4 inhibits the binding of PHR2 to its cis-element and reduces the targeting of PHR2 to the nucleus. However, when plants grow under Pi deficiency, the degradation of SPX4 is accelerated through the 26S proteasome pathway, thereby releasing PHR2 into the nucleus and activating the expression of PSI genes. Because the level of SPX4 is responsive to Pi concentration and SPX4 interacts with PHR2 and regulates its activity, this suggests that SPX4 senses the internal Pi concentration under diverse Pi conditions and regulates appropriate responses to maintain Pi homeostasis in plants.

Natural variation of GmRj2/Rfg1 determines symbiont differentiation in soybean.

Symbiotic nitrogen fixation (SNF) provides much of the N utilized by leguminous plants throughout growth and development. Legumes may simultaneously establish symbiosis with different taxa of microbial symbionts. Yet, the mechanisms used to steer associations toward symbionts that are most propitious across variations in soil types remain mysterious. Here, we demonstrate that GmRj2/Rfg1 is responsible for regulating symbiosis with multiple taxa of soybean symbionts. In our experiments, the GmRj2/Rfg1SC haplotype favored association with Bradyrhizobia, which is mostly distributed in acid soils, whereas the GmRj2/Rfg1HH haplotype and knockout mutants of GmRj2/Rfg1SC associated equally with Bradyrhizobia and Sinorhizobium. Association between GmRj2/Rfg1 and NopP, furthermore, appeared to be involved in symbiont selection. Furthermore, geographic distribution analysis of 1,821 soybean accessions showed that GmRj2/Rfg1SC haplotypes were enriched in acidic soils where Bradyrhizobia were the dominant symbionts, whereas GmRj2/Rfg1HH haplotypes were most prevalent in alkaline soils dominated by Sinorhizobium, and neutral soils harbored no apparent predilections toward either haplotype. Taken together, our results suggest that GmRj2/Rfg1 regulates symbiosis with different symbionts and is a strong determinant of soybean adaptability across soil regions. As a consequence, the manipulation of the GmRj2/Rfg1 genotype or application of suitable symbionts according to the haplotype at the GmRj2/Rfg1 locus might be suitable strategies to explore for increasing soybean yield through the management of SNF.

Expression and Functional Study of BcWRKY1 in Baphicacanthus cusia (Nees) Bremek.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an important medicinal plant. Its effective substances including indigo and indirubin are metabolites in indoleacetate metabolic pathway. Based on a previous transcriptome sequencing analysis, a WRKY transcription factor, BcWRKY1, in B. cusia was identified, showing significant correlation with effective substances from B. cusia. In this study, BcWRKY1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the BcWRKY1 gene was 916 bp in length, containing three exons and two introns. The open reading frame (ORF) of BcWRKY1 was 534 bp in length and encoded a WRKY domain-containing protein with 177 amino acids residues. Subcellular localization showed that BcWRKY1 protein was mainly localized in the nucleus. It could bind to the W-box motif and its role in transcriptional activation was confirmed in yeast. The function of BcWRKY1 was investigated by overexpressing BcWRKY1 in Arabidopsis thaliana. Metabolic profiles in wild type and BcWRKY1-OX1 transgenic Arabidopsis thaliana were analyzed with LC-MS. Results showed that the metabolic profile was significantly changed in BcWRKY1-OX1 transgenic Arabidopsis thaliana compared with wild type. Furthermore, indole-related metabolites were significantly increased in BcWRKY1-OX1 transgenic Arabidopsis thaliana, and the metabolic pathway analysis showed that flavonoid biosynthesis was significantly enriched. Overexpression of BcWRKY1 significantly changed flavonoid and indole metabolism and indole-related metabolites were significantly upregulated. We postulated that the BcWRKY1 transcription factor might be involved in the regulation of effective substances metabolism in B. cusia.

Functional Investigation of Plant Growth Promoting Rhizobacterial Communities in Sugarcane.

Plant microbiota are of great importance for host nutrition and health. As a C4 plant species with a high carbon fixation capacity, sugarcane also associates with beneficial microbes, though mechanisms underlying sugarcane root-associated community development remain unclear. Here, we identify microbes that are specifically enriched around sugarcane roots and report results of functional testing of potentially beneficial microbes propagating with sugarcane plants. First, we analyzed recruitment of microbes through analysis of 16S rDNA enrichment in greenhouse cultured sugarcane seedlings growing in field soil. Then, plant-associated microbes were isolated and assayed for beneficial activity, first in greenhouse experiments, followed by field trials for selected microbial strains. The promising beneficial microbe SRB-109, which quickly colonized both roots and shoots of sugarcane plants, significantly promoted sugarcane growth in field trials, nitrogen and potassium acquisition increasing by 35.68 and 28.35%, respectively. Taken together, this report demonstrates successful identification and utilization of beneficial plant-associated microbes in sugarcane production. Further development might facilitate incorporation of such growth-promoting microbial applications in large-scale sugarcane production, which may not only increase yields but also reduce fertilizer costs and runoff.

Rhizobium Inoculation Drives the Shifting of Rhizosphere Fungal Community in a Host Genotype Dependent Manner.

Rhizosphere microorganisms play important roles in plant health and nutrition, and interactions among plants and microorganisms are important for establishment of root microbiomes. As yet, plant-microbe and microbe-microbe interactions in the rhizosphere remain largely mysterious. In this study, rhizosphere fungal community structure was first studied in a field experiment with two soybean cultivars contrasting in nodulation grown in two rhizobium inoculation treatments. Following this, recombinant inbred lines (RILs) contrasting in markers across three QTLs for biological nitrogen fixation (BNF) were evaluated for effects of genotype and rhizobium inoculation to the rhizosphere fungal community as assessed using ITS1 amplicon sequencing. The soybean plants tested herein not only hosted rhizosphere fungal communities that were distinct from bulk soils, but also specifically recruited and enriched Cladosporium from bulk soils. The resulting rhizosphere fungal communities varied among soybean genotypes, as well as, between rhizobium inoculation treatments. Besides, Cladosporium were mostly enriched in the rhizospheres of soybean genotypes carrying two or three favorable BNF QTLs, suggesting a close association between soybean traits associated with nodulation and those affecting the rhizosphere fungal community. This inference was bolstered by the observation that introduction of exogenous rhizobia significantly altered rhizosphere fungal communities to the point that these communities could be distinguished based on the combination of soybean genotype and whether exogenous rhizobia was applied. Interestingly, grouping of host plants by BNF QTLs also distinguished fungal community responses to rhizobium inoculation. Taken together, these results reveal that complex cross-kingdom interactions exist among host plants, symbiotic N2 fixing bacteria and fungal communities in the soybean rhizosphere.

Deciphering the bacterial composition in the rhizosphere of Baphicacanthus cusia (NeeS) Bremek.

Rhizobacteria is an important ingredient for growth and health of medicinal herbs, and synthesis of pharmacological effective substances from it. In this study, we investigated the community structure and composition of rhizobacteria in Baphicacanthus cusia (NeeS) Bremek via 16S rRNA amplicon sequencing. We obtained an average of 3,371 and 3,730 OTUs for bulk soil and rhizosphere soil samples respectively. Beta diversity analysis suggested that the bacterial community in the rhizosphere was distinctive from that in the bulk soil, which indicates that B.cusia can specifically recruit microbes from bulk soil and host in the rhizosphere. Burkholderia was significantly enriched in the rhizosphere. Burkholderia is a potentially beneficial bacteria that has been reported to play a major role in the synthesis of indigo, which was a major effective substances in B. cusia. In addition, we found that Bacilli were depleted in the rhizosphere, which are useful for biocontrol of soil-borne diseases, and this may explain the continuous cropping obstacles in B. cusia. Our results revealed the structure and composition of bacterial diversity in B. cusia rhizosphere, and provided clues for improving the medicinal value of B. cusia in the future.

SPX proteins regulate Pi homeostasis and signaling in different subcellular level.

To cope with low phosphate (Pi) availability, plants have to adjust its gene expression profile to facilitate Pi acquisition and remobilization. Sensing the levels of Pi is essential for reprogramming the gene expression profile to adapt to the fluctuating Pi environment. AtPHR1 in Arabidopsis and OsPHR2 in rice are central regulators of Pi signaling, which regulates the expression of phosphate starvation-induced (PSI) genes by binding to the P1BS elements in the promoter of PSI genes. However, how the Pi level affects the central regulator to regulate the PSI genes have puzzled us for a decade. Recent progress in SPX proteins indicated that the SPX proteins play important role in regulating the activity of central regulator AtPHR1/OsPHR2 in a Pi dependent manner at different subcellular levels.