Coordinated responses of leaf and nodule traits contribute to the accumulation of N in relay intercropped soybean

Maize(Zea mays L.)-soybean(Glycine max L.Merr.)relay intercropping provides a way to enhance land productivity.However,the late-planted soybean suffers from shading by the maize.After maize harvest,how the recovery growth influences the leaf and nodule traits remains unclear.A three-year field experiment was conducted to evaluate the effects of genotypes,i.e.,supernodulating(nts1007),Nandou 12(ND12),and Guixia 3(GX3),and crop configurations,i.e.,the interspecific row spacing of 45(I45),60(I60),75 cm(I75),and sole soybean(SS),on soybean recovery growth and N fixation.The results showed that intercropping reduced the soybean total leaf area(LA)by reducing both the leaf number(LN)and unit leaflet area(LUA),and it reduced the nodule dry weight(NW)by reducing both the nodule number(NN)and nodule diameter(ND)compared with the SS.The correlation and principal component analysis(PCA)indicated a co-variability of the leaf and nodule traits in response to the genotype and crop configuration interactions.During the recovery growth stages,the compensatory growth promoted soybean growth to reduce the gaps of leaf and nodule traits between intercropping and SS.The relative growth rates of ureide(RGR_U)and nitrogen(RGR_N)accumulation were higher in intercropping than in SS.Intercropping achieved more significant sucrose and starch contents compared with SS.ND12 and GX3 showed more robust compensatory growth than nts1007 in intercropping.Although the recovery growth of relay intercropping soybean improved biomass and nitrogen accumulation,ND12 gained a more significant partial land equivalent ratio(pLER)than GX3.The I60 treatment achieved more robust compensation effects on biomass and N accumulation than the other configurations.Meanwhile,I60 showed a higher nodule sucrose content and greater shoot ureide and N accumulation than SS.Finally,intercropping ND12 with maize using an interspecific row spacing of 60 cm was optimal for both yield advantage and N accumulation.

Identification of Rhizobia Isolated from Nodules of Mexican Commercial Soybean Varieties

Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybean varieties in high-production regions. Multilocus sequence analysis (MLSA) was employed for enhanced species resolution. The study identified six Bradyrhizobium species: Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 6, Bradyrhizobium elkanii USDA 76, Bradyrhizobium neotropicale, Bradyrhizobium lablabi, and Bradyrhizobium icense. Bradyrhizobium japonicum USDA 110 predominated in the soils, displaying symbiotic preference for the Huasteca 400 variety. However, phylogenetic analysis didn't reveal a clear association between strains, soil, and soybean variety. This research sheds light on the diversity of rhizobia in Mexican soybean cultivation, contributing to the understanding of symbiotic relationships in soybean production systems.

GmNLP7a inhibits soybean nodulation by interacting with GmNIN1a

Nitrogen(N) is an essential macronutrient for plant growth and productivity. Leguminous plants establish symbiotic relationships with nitrogen-fixing rhizobial bacteria to use atmospheric dinitrogen gas to meet high N demand under low-N conditions. Nodule formation and N fixation are energy-consuming processes and are inhibited by nitrate present in the environment. Previous studies in model leguminous plants characterized NIN-LIKE PROTEIN(NLP) proteins that mediate nitrate control of root nodule symbiosis, but the mechanism by which nitrate regulates soybean root nodules via NLP remains unclear. In the soybean genome we found four homologs of AtNLP7, named GmNLP7a–GmNLP7d. We showed that the expression of GmNLP7s is responsive to nitrate but not to rhizobial infection and localized GmNLP7a to the nucleus. Downregulation of GmNLP7s increased nodule number, and overexpression of GmNLP7a(GmNLP7aOE) reduced nodule number regardless of nitrate availability, suggesting a negative role for GmNLP7s in nodulation. Nitrogenase activity in the GmNLP7aOE line was comparable to that of the wild type, indicating that GmNLP7a does not affect mature nodule activity. Overexpression of GmNLP7a downregulated the expression of GmNIN1a and GmENOD40-1. GmNLP7a interacted with GmNIN1a via the PB1domain. Our results reveal a new regulator of GmNLP7 in nodulation and a molecular mechanism by which nitrate affects nodule number in soybean.

Isolation of Soybean Nodule Bacteria and Nodule Formation in Uzbekistan Soybean Varieties

The main goal of our research work is to search for nodule bacteria of local soybean varieties and to identify and study their nodule-forming properties. In the present study, soil samples from fields in the Tashkent, Andijan, Bukhara, Jizzakh, Kashkadarya, Navoi, Namangan, Samarkand, Surkhandarya, Syrdarya, Fergana, and Khorezm regions of Uzbekistan were studied for the formation of symbiotic nodules in local soybean varieties. Nodules formed only in the soils of the Tashkent region in the root systems of local soybean varieties (Madad, Sevinch, Dostlik, Parvoz, Gavkhar, Khasildar, Baraka, Tashkent, Uzbekistan-6, Tumaris, Nafis, Orzu) were formed from 22 to 40 nodule. Forty-one bacterial species belonging to the genus Bradyrhizobium were isolated from the nodules of the different varieties. The specificity, virulence, and symbiotic efficacy of 12 active nodule bacteria were compared in the local Madad, Sevinch, Dostlik, and Parvoz varieties. The root systems of these varieties formed from 2 to 14 symbiotic pink nodules 0.5 - 10 mm in size. Inoculation of the Madad and Dostlik varieties with their specific M5-1 and D24-1 nodule bacteria resulted in a symbiotic efficiency 46.6% - 54.4% higher than in uninoculated control plants. Notably, the foreign inoculum “Rizovit” (Kazakhstan), created on the basis of Bradyrhizobium japonicum, did not form any nodules on the roots of the local Uzbekistan varieties. The main reason for this may be the difference in the genetic origin of foreign soybean varieties and domestic Uzbekistan varieties. The nucleotide sequences of 16S rRNA genes of nodule bacteria M5-1, S7-2, D24-1, and P12-1 showed 97.07% similarity with the 16S rRNA genes of Bradyrhizobium japonicum PRY65 (AF239848.2) and 98.98% similarity with Bradyrhizobium japonicum PRY62 (AF239847.2).

Morpho-Physiological and Biochemical Characterization of Soybean-Associated Rhizobia and Effect of Their Liquid Inoculant Formulation on Nodulation of Host Plants in the Cameroon Cotton Fields Zone

The aim of this research was to assess the diversity of the Cameroon cotton zone in soybean associated rhizobia in order to formulate the most efficient elite inoculant to boost both the cotton and soybean production. Therefore, soybean associated rhizobia were isolated and characterized morphologically, physiologically and biochemically on YEMA culture media. For each of the two soybean varieties (Houla1 and TGX1910 14F) used, the trials were laid out in two IRAD-fields of North Cameroon (Sanguere-Paul) and Far-North (Soukoundou) respectively, under a complete randomized complete block design, the isolate formulations representing the treatments. The six isolated strains (IS1, IS2, IS3, IS4, IS5, IS6) from which seven liquid inoculant were formulated were revealed to belong to the same slow growing group of rhizobia, with a high level of tolerance to temperature, pH, and salinity, with optimum growth at respectively 28˚C, pH (7 - 9), salt (1% - 5%). Not surprisingly, root nodules were formed by both inoculated and uninoculated soybean plants. However, the most efficient soybean-rhizobia symbiosis for nodulations were isolate IS6 associated to TGX1910 14F variety, and isolate IS5 associated to Houla1variety at Sanguere-Paul. Whereas isolate M was associated to TGX1910 14F variety, Houla 1 variety had affinity with native rhizobia isolates at Soukoundou. The present results suggest the adaptability of rhizobia isolates to a particular soybean variety at a particular cotton fields zone. These findings should be taken into consideration for commercial inoculant formulation.

GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean(Glycine max [L.] Merr.)

As an important food crop and oil crop, soybean(Glycine max [L.] Merr.) is capable of nitrogen-fixing by root nodule. Previous studies showed that GmNMH7, a transcription factor of MADS-box family, is associated with nodule development, but its specific function remained unknown. In this study, we found that GmN MH7 was specifically expressed in root and nodule and the expression pattern of GmNMH7 was similar to several genes involved in early development of nodule(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) after rhizobia inoculation. The earlier expression peak of GmNMH7 compared to the other genes(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) indicated that the gene is related to the nod factor(NF) signaling pathway and functions at the early development of nodule. Over-expression of GmNMH7 in hairy roots significantly reduced the nodule number and the root length. In the transgenic hairy roots, overexpression of GmN MH7 significantly down-regulated the expression levels of GmE NOD40-1, GmE NOD40-2, and GmN FR5α. Moreover, the expression of GmNMH7 could respond to abscisic acid(ABA) and gibberellin(GA_3) treatment in the root of Zigongdongdou seedlings. Over-expressing GmNMH7 gene reduced the content of ABA, and increased the content of GA_3 in the positive transgenic hairy roots. Therefore, we concluded that GmNMH7 might participate in the NF signaling pathway and negatively regulate nodulation probably through regulating the content of GA_3.

Nodule Formation and Development in Soybeans(Glycine max L.) in Response to Phosphorus Supply in Solution Culture

Phosphorus (P) is necessary for growth and nitrogen fixation, and thus its deficiency is a major factor limiting legume production in most agricultural soils. The effect of phosphorus supply on nodule development and its role in soybeans (Glycine max L.) was studied in a nutrient solution. Plants were inoculated with Bradyrhizobium japonicum and grown for 35 days in a glasshouse at a day and night temperature of 25℃and 15℃, respectively. Although increasing P supply increased the concentrations of P and N in the shoots and roots, the external P supply did not significantly affect the P concentration in the nodules, and the N fixed per unit nodule biomass decreased with increasing P supply. The nitrogen content in the shoots correlated well with the P content (r = 0.92**). At an inoculation level of 102 cells mL-1, the P supply did not affect the number of nodules; however, at inoculation levels of 103.5 and 105 cells mL-1, increasing P supply increased both the number and size of nodules. Irrespective of the inoculation level, increasing P supply increased the nodule biomass relative to the biomass of the host plant. It is suggested that the P deficiency specifically inhibited the nodule development and thereby the total N2 fixation.

Specific Expression of a Novel Nodulin GmN479 Gene in the Infected Cells of Soybean (Glycine max) Nodules

A novel nodulin gene, GmN479 genomic clone composing of 3 630 nucleotides was isolated from mature soybean nodules using GmN479 cDNA as a probe by subtractive hybridization procedure. GmN479 encodes 170 amino acids with 2.09 kb nucleotides promoter region, and contains two important upstream promoter elements, one is a conserved cis-acting sequence motif 5′-AAAGAT-3′ controlling nodulin gene expression, and the other is typical CAAT boxes. GmN479 gene has a single zinc-finger C2H2 domain YSCAFCQRGFSNAQALLGGHMNIH and a conserved motif, QALGGHMN in the zinc-finger with a short leucine repeat in the LDLELRLGL motif closed to C-terminal. These two conserved motifs share respectively higher identity with those in the floral regulator SUPERMAN gene, indicating that GmN479 may function as a transcriptional regulator, and is a likely candidate for playing a role in nodule-morphogenesis. Blotting data showed that GmN479 is a single copy presenting in the genome of soybean nodule, and its expression profile is similar to that of Lb-a, but it is different from that of ENOD2. GUS staining showed that GmN479 promoter just functions in the infected cells of nodules, indicating that the GmN479 is one of the truly symbiotically induced host genes, and belongs to a late nodulin gene. The expression pattern of GmN479 gene seems to imply that it may be closely related to the development of the nodule. In a sense, it may be a useful marker for identifying the development of the infected cell system in the nodules of soybean.

Incompatible Nodulation of <i>Bradyrhizobium elkanii</i>Strains BLY3-8 and BLY6-1 with <i>Rj</i>3 Gene-Harboring Soybean Cultivars

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some Bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain Bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

Characterization of chromosome segment substitution lines reveals candidate genes associated with the nodule number in soybean

Soybean is one of the most important food crops worldwide.Like other legumes,soybean can form symbiotic relationships with Rhizobium species.Nitrogen fixation of soybean via its symbiosis with Rhizobium is pivotal for sustainable agriculture.Type Ⅲ effectors(T3Es)are essential regulators of the establishment of the symbiosis,and nodule number is a feature of nitrogen-affected nodulation.However,genes encoding T3Es at quantitative trait loci(QTLs)related to nodulation have rarely been identified.Chromosome segment substitution lines(CSSLs)have a common genetic background but only a few loci with heterogeneous genetic information;thus,they are suitable materials for identifying candidate genes at a target locus.In this study,a CSSL population was used to identify the QTLs related to nodule number in soybean.Single nucleotide polymorphism(SNP)markers and candidate genes within the QTLs interval were detected,and it was determined which genes showed differential expression between isolines.Four candidate genes(GmCDPK28,GmNAC1,GmbHLH,and GmERF5)linked to the SNPs were identified as being related to nodule traits and pivotal processes and pathways involved in symbiosis establishment.A candidate gene(GmERF5)encoding a transcription factor that may interact directly with the T3E NopAA was identified.The confirmed CSSLs with important segments and candidate genes identified in this study are valuable resources for further studies on the genetic network and T3Es involved in the signaling pathway that is essential for symbiosis establishment.

Soybean Nodulation and Plant Response to Nitrogen and Sulfur Fertilization in the Northern US

Soybean [Glycine max (L.) Merrill] seed yields in the northern United States may increase with the application of fertilizers;however Nitrogen (N) may decrease root nodulation. This study was conducted to understand the impact of N and sulfur (S) fertilization on soybean nodulation, plant, shoot and root biomass. Two cultivars were planted in experiments across ten site-years during 2015-2016. Plant observations took place at the V4 and R4 soybean growth stages. There were 41% more nodules per plant at R4 compared to V4 (38.3 vs 27.2 nodules, respectively). Cultivars responded differently to N and S fertilizer. The nodules per plant between the cultivars (30.3 vs 24.4) were different as well as the percent medium and large-sized nodules, which indicates the need to evaluate additional genotypes. Adding N decreased root nodulation (from 31.8 to 23.7 nodules per plant) and decreased nodule size but had no effect on plant, shoot or root mass. Averaged across N rates total plant mass was 2.26 and 11.36 g per plant at V4 and R4, respectively. Shoot mass, average across N rates was 1.77 and 9.65 g per plant at V4 and R4, respectively, and root mass, average across N rates was 0.49 and 1.71 g per plant at V4 and R4, respectively. Sulfur did not have an effect on nodules per plant but increased the percent medium size nodules at the R4 observation. There was no N by S interaction observed for nodule number, size of the nodules, and plant, root and shoot mass. As cultivars differed in their nodulation response to N and S, additional research would be helpful to screen other cultivars.

Nodulation Capacity of Argentinean Soybean (<i>Glycine max</i>L. Merr) Cultivars Inoculated with Commercial Strains of <i>Bradyrhizobium japonicum</i>

The purpose of this research was to evaluate the nodulation potential of 31 Argentinean soybean commercial cultivars. Those with the highest nodulation capacity response developed twice the amount of nodules than the low nodulating ones, which is the variation contained in soybean genotypes. Furthermore, this was not due to bacterial promiscuity, since the response was independent of the bradyrhizobia strain inoculated. The ability of cultivars to develop a larger number and biomass of nodules was unrelated with the maturity group they belong to and also was not a response to quorum sensing effects. Our results suggest that breeding programs can be aimed at improving the nodulation capacity of soybean and that cultivars from different maturity groups can be a source of nodulation QTLs.

多组学分析磷胁迫下大豆根瘤碳氮代谢途径

为解析磷胁迫对大豆根瘤固氮作用的影响,对磷胁迫处理15 d的大豆根瘤进行代谢组学和蛋白质组学检测,分析根瘤中差异代谢物质和差异蛋白及参与的代谢通路。结果表明:大豆根瘤中代谢物质和蛋白上调和下调以响应磷胁迫,参与11个碳代谢途径和19个氮代谢途径,糖类物质下调,参与糖酵解、果糖和甘露糖代谢、半乳糖代谢、淀粉和蔗糖代谢等碳代谢途径,导致根瘤中能量供应不足,进而影响根瘤固氮作用;氨基酸和酶以上调为主,富集在氨基酸代谢、氮代谢、氨酰tRNA生物合成及嘌呤代谢通路,是大豆根瘤响应磷胁迫的重要氮代谢途径;氨基酸上调为TCA循环中间产物提供碳骨架,碳氮代谢协同作用响应磷胁迫对大豆根瘤固氮作用的影响。研究结果为解析磷胁迫影响大豆根瘤固氮作用的生理机制提供理论参考。

冀北坝上大豆结瘤相关性状全基因组关联分析

【目的】挖掘适应坝上生态条件的高效结瘤大豆种质,鉴定调控大豆-根瘤菌共生结瘤的遗传位点和候选基因,提高大豆共生固氮效率。【方法】以包含260份大豆种质的自然群体为研究对象,在河北坝上室外盆栽条件下接种根瘤菌菌株USDA110。以单株根瘤数量、单株根瘤干重数值作为表型数据,结合大豆260份种质基因型数据,对其进行全基因组关联分析,挖掘大豆共生结瘤相关基因。【结果】共检测到18个与大豆根瘤数量显著关联的SNP,分别位于第2、7、8、13、18和19染色体上。其中,位于第2染色体上的显著关联位点BARC_2.01_Chr02_43161654_A_G是控制大豆根瘤数量的主效位点(LOD=3.89),对该位点上下游共200 kb区间进行连锁不平衡分析,在包含BARC_2.01_Chr02_43161654_A_G的连锁区间内,共获得10个调控大豆根瘤数量候选基因,通过单倍型分析发现,Glyma.02G243200不同单倍型所对应的材料之间的根瘤数量具有显著差异(P<0.05),在SoyBase数据库中查询该基因的表达模式发现,Glyma.02G243200在根毛中表达。该基因可能是影响大豆根瘤数量的关键基因。共检测到6个与大豆根瘤干重显著关联的SNP,分别位于第6、18和20染色体上。其中,位于第6染色体上的显著关联位点BARC_2.01_Chr06_6069381_G_A和BARC_2.01_Chr06_6192925_T_C是控制大豆根瘤干重的主效位点(LOD=3.49和LOD=3.35),对BARC_2.01_Chr06_6069381_G_A上游100 kb和BARC_2.01_Chr06_6192925_T_C下游100 kb区间进行连锁不平衡分析,获得14个调控大豆根瘤干重候选基因,并进行单倍型分析。其中,Glyma.06G079600和Glyma.06G079900不同单倍型所对应的材料之间的根瘤干重具有显著差异(P<0.01,P<0.001),在SoyBase数据库中查询这两个基因的表达模式发现,Glyma.06G079600和Glyma.06G079900在根中表达。这两个基因可能是影响大豆根瘤干重的关键基因。【结论】在第2染色体上发现一个与根瘤数量显著相关的候选基因,在第6染色体上发现2个与根瘤干重显著相关的候选基因。

大豆根瘤菌Y63菌株的分离与鉴定

在大豆生产中充分发挥共生固氮作用,在减少氮肥的施用、增加土壤微生物的活性、保证耕地及农业生态系统健康、促进绿色农业的可持续性发展等方面具有重要意义。本研究以高产、优质、多抗、广适的大豆品种中豆63为捕获宿主,对湖北省洪湖市高产示范田中的中豆63根瘤菌进行分离与鉴定。采用平板划线分离、BTB反应试验等方法筛选出符合根瘤菌表观特征的菌株,并将获得的菌株进行16S rDNA序列扩增,构建系统发育树,鉴定从中豆63的单个根瘤中分离得到的2个大豆根瘤菌菌株,分别命名为Y63-1与Y63-2。通过抗生素抗性分析表明,Y63-1具有羧苄青霉素(Carbenicillin)、头孢霉素(Cefotaximesodium)、氨苄青霉素(Ampicillin)和利福平(Rifampin)4种抗性,Y63-2具有硫酸卡那霉素(Kanamycin)、羧苄青霉素(Carbenicillin)、氨苄青霉素(Ampicillin)和利福平(Rifampin)4种抗性。最后以慢生型根瘤菌113-2及快生型根瘤菌HH103作为对照,将获得的两个根瘤菌Y63-1与Y63-2按一定的比例通过盆栽回接中豆63,在接种后42天及51天分别对不同接种比例下中豆63植株地上部分鲜重、结瘤数及根瘤干重进行统计学分析,结果表明Y63-1∶Y63-2=2∶1接种中豆63时结瘤效应最佳。混合接种后根瘤中Y63-1与Y63-2的比例初步鉴定结果显示,Y63-1为主效根瘤菌。本研究丰富了我国的根瘤菌资源,研究结果为大豆根瘤菌混合接种技术在大豆生产中的推广应用提供了理论依据,也为生产中通过接种高效根瘤菌提升中豆63及其它品种产量潜力奠定了理论基础。

大豆miR10193家族成员鉴定及其结瘤相关表达分析

miR10193是在大豆中和线虫侵染等相关的一个miRNA家族,其在大豆共生结瘤中的作用仍不清楚。为系统鉴定大豆miR10193家族成员并分析其在共生固氮过程中的表达模式,本研究在全基因组水平上鉴定大豆miR10193家族成员并进行生物信息学分析,采用qRT-PCR方法分析其在不同大豆组织和根瘤菌侵染早期的表达模式。结果显示:共鉴定出12个miR10193成员,分别位于第3、4、7、8、10、13、16、19和20号染色体上。miR10193家族成员成熟序列和星标序列高度保守,它们的前体序列均可生成稳定的二级结构。miR10193家族前体序列的进化分析可将miR10193家族成员分为两个亚家族,其中gma-miR10193c和gma-miR10193h存在串联复制。顺式调控元件分析发现该家族成员可能参与植物激素、胁迫、生长和发育等过程。miR10193家族成员在不同组织的表达量分析结果表明,多个miR10193家族成员在根瘤中表达最高,表明其可能参与大豆共生结瘤的调控。除gma-miR10193d/j/k/l外,其它miR10193家族成员均在根瘤中高表达,并发现miR10193家族成员均显著响应根瘤菌的侵染。靶基因预测表明BTB/POZ结构域蛋白和2OG-Fe(Ⅱ)氧化酶家族蛋白可能是miR10193的靶基因。本研究为研究大豆miR10193家族及其在豆科结瘤固氮过程中的功能和机制提供了理论基础。

短期低温对大豆苗期生长和结瘤固氮的影响

采用早熟大豆品种“黑河43”、晚熟大豆品种“东农53”进行盆栽试验,出苗后进行连续7d的低温处理(LT),昼夜温度设定为13℃/3℃,以25℃/10℃为对照,从大豆出苗28d开始,每7d进行破坏性取样共4次,测定地上部和根干物质量、根瘤生物量、全氮含量及根瘤固氮量,研究苗期短期低温对大豆生长、结瘤和固氮能力的影响。结果表明:(1)大豆出苗后7d短期低温胁迫会延缓大豆生长发育。出苗后28~42d大豆生物量显著低于CK处理,出苗后49d低温胁迫对两个大豆品种生物量均无显著影响。(2)与CK相比,低温对“东农53”和“黑河43”根瘤形成的显著抑制作用分别出现在苗后42d和35d,根瘤数量分别降低了58.8%和72.0%。出苗后49d,低温使“东农53”和“黑河43”根瘤干重分别减少48.9%和48.5%。(3)低温刺激了两个品种大豆的生物固氮能力。在出苗后49d,低温处理“东农53”和“黑河43”的生物固氮量、单位根瘤生物量的固氮量和单个根瘤的固氮量分别增加了89.9%、118.9%,249.3%、172.6%,150.6%、114.2%。生物固氮百分率分别增加了26.4个和24.5个百分点。综合来看,大豆出苗后遭遇低温冷害会延缓生长发育,并抑制根瘤的形成与生长。低温胁迫解除后大豆生长逐步恢复。短期低温对大豆根瘤固氮量和根瘤固氮能力有刺激作用,对早熟品种“黑河43”刺激作用更明显。为了更好地适应东北大豆产区早春低温环境,推荐选用早熟品种大豆。

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process

The soybean root system is complex.In addition to being composed of various cell types,the soybean root system includes the primary root,the lateral roots,and the nodule,an organ in which mutualistic symbiosis with N-fixing rhizobia occurs.A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont,resulting from the close cooperation between the plant cell and the bacteria.To date,the transcriptome of individual cells isolated from developing soybean nodules has been established,but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized.Using single-nucleus RNA-seq and Molecular Cartography technologies,we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B.diazoefficiens-infected cells in the mature soybean nodule,including those actively involved in nitrogen fixation and those engaged in senescence.Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process.For instance,we functionally characterized the role of GmFWL3,a plasma membrane microdomain-associated protein that controls rhizobial infection.Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

大豆蛋白磷酸酶基因GmPP2C28的克隆与表达分析

为解析植物蛋白磷酸酶2C(PP2C)在豆科植物与根瘤菌共生互作过程中的作用,以大豆为研究材料,克隆了大豆的一个蛋白磷酸酶基因GmPP2C28,并对其编码蛋白进行了亚细胞定位,随后在大肠杆菌中对其重组蛋白进行了表达和纯化。此外,通过荧光定量PCR技术,分析了GmPP2C28基因在大豆不同组织及接种根瘤菌后不同时期根系中的表达模式。结果表明,GmPP2C28完整编码区的cDNA序列长度为1029 bp,编码343个氨基酸,其编码蛋白定位于拟南芥原生质体的细胞核。体外重组蛋白以可溶形式高效表达,目的条带大小为37 kD左右。GmPP2C28基因在大豆的根和根瘤中表达水平较高,在接种根瘤菌后的大豆根中表达水平呈现先升高再下降的表达模式。

GmACP expression is decreased in Gm NORK knockdown transgenic soybean roots

NORK and soybean acyl carrier protein(ACP) both play important roles in nodulation.However,the relationship between Nod factor signaling and fatty acid(FA) biosynthesis during symbiotic development is unknown.In this study,an RNAi plasmid of Gm NORK was constructed and transformed into soybean roots by Agrobacterium rhizogene-mediated hairy-root transformation.The nodule number decreased substantially in Gm NORK knockdown soybean transgenic roots.To investigate the relationship between Gm ACP and Nod factor signaling,we measured Gm ACP expression levels in Gm NORK RNAi soybean transgenic roots and found that rhizobia inoculation led to substantially reduced Gm ACP expression.Thus,FA biosynthesis was affected by Nod factor signaling during nodule development in soybean,a finding that provides valuable information that improves our understanding of the functions of Gm NORK and Gm ACP in symbiotic signaling and nodule development.