Dissection of genetic overlap of drought and low-temperature tolerance QTLs at the germination stage using backcross introgression lines in soybean

Northeast of China is the main soybean production area, drought and low-temperature tolerance are both main factors involved in reducing soybean yield and limiting planting regions, the most effective way to solve this problem is to breed cultivars with drought and low-temperature tolerance. A set of the BC₂F₃ lines was constructed with Hongfeng 11 as recurrent parent and Harosoy as donor parent, and screened in drought and low-temperature condition at the germination stage. Related QTLs were obtained by Chi-test and ANOVA analysis with genotypic and phenotypic data. Eighteen QTLs of drought tolerance and 23 QTLs of low-temperature tolerance were detected. Among them, 12 QTLs were correlated with both drought and low-temperature tolerance, which showed a partial genetic overlap between drought and low-temperature tolerance at the germination stage in soybean. Among the 12 genetic overlap QTLs, Satt253, Satt513, Satt693, Satt240, Satt323, and Satt255 were detected by at least one method for both drought and low-temperature tolerance. Satt557, Satt452, Sat_331, Satt338, Satt271, and Satt588 were detected by only one analysis method. The QTLs detected above were significant loci for drought or low-temperature tolerance in soybean. This will play an important role in MAS for development of both drought and low-temperature tolerance variety.     

大豆种质资源萌发期耐旱性评价

随着全球气候的变化,干旱已成为限制大豆产量的重要环境因素之一,大豆种质资源耐旱性鉴定对大豆耐旱品种的培育及大豆耐旱机理的研究具有重要的意义。本研究首先选择4份耐旱性不同的材料,以PEG-6000作为渗透胁迫剂,研究了大豆种质在0%、10%、15%、16%、18%、20%、30%的PEG-6000溶液的胁迫下,相对发芽率、相对发芽势、相对胚根长度、萌发耐旱指数的变化。结果表明15%~18%PEG-6000溶液是大豆萌发期耐旱鉴定的适宜浓度范围。本研究采用16%PEG-6000溶液对568份大豆资源进行大豆种子萌发期耐旱鉴定,以相对发芽势、相对发芽率、相对胚根长度、相对苗高、萌发耐旱指数、活力指数作为评价指标,应用平均隶属函数评价法对大豆种质萌发期耐旱性进行综合评价,筛选出耐旱种质4份,较耐旱种质18份,中间型种质110份,较敏感种质194份,敏感型种质242份。     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

Detection of Quantitative Trait Loci for Yield and Drought Tolerance Traits in Soybean Using a Recombinant Inbred Line Population

To investigate the genetic basis of drought tolerance in soybean ( Glycine max L. Merr.) a recombinant inbred population with 184 F_2:7:11 lines developed from a cross between Kefeng1 (drought tolerant) and Nannong1138-2 (drought sensitive) were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci (QTLs) were identified: 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and D1b, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.     

Metabolic and Physiological Changes Induced by Nitric Oxide and Its Impact on Drought Tolerance in Soybean

Nitric oxide (NO) is an important signaling molecule that plays a pivotal role in stress tolerance. To study the role of NO in drought tolerance and elucidate the underlying mechanisms, NO (0 and 100 μM) was applied to drought-treated soybean plants. Drought stress was imposed by PEG (5% (W/V) of PEG 6000. Nitric oxide improved growth of soybean plants under drought as evidenced by enhanced dry weight (30%). Nitric oxide caused a remarkable increase in activities of catalase and superoxide dismutase (SOD) and SOD expression (14.8-fold), which led to a significant decline in malondealdehyde content under drought conditions. Nitric oxide induced proline biosynthesis due to enhancing pyrroline-5- carboxylate synthetase (P5CS) expression (43.66-fold). The growth-promoting effect of NO application in soybean plants was concomitant with change in metabolic profile (phenolic acid and flavonoid compounds). Nitric oxide up-regulated of phenylalanine ammonia-lyase (PAL) expression in drought-treated plants and may influence on the phenylpropanoid production. Nitric oxide increased salicylic acid (SA) content in soybean plants under stress. So, NO and SA are jointly responsible for boosted tolerance to drought stress in soybean plants. The decrease in unsaturated fatty acid through NO application might reflect a reduction in oxidative damage. These results propose a multifaceted contribution of NO through regulation of physiological and metabolic processes in response to drought stress.

     

Separately and simultaneously induced dark chilling and drought stress effects on photosynthesis, proline accumulation and antioxidant metabolism in soybean

The effects of separately or simultaneously induced dark chilling and drought stress were evaluated in two Glycine max (L.) Merrill cultivars. For the separately induced dark chilling treatment (C), plants were incubated at 8 °C during 9 consecutive dark periods. During the days, plants were kept at normal growth temperatures. For the separately induced drought treatment (D), plants were maintained at normal growth temperatures without irrigation. For the simultaneously induced dark chilling and drought stress treatment (CD), plants were dark chilled without irrigation. All treatments caused similar decreases in pre-dawn leaf water potential, but resulted in distinct physiological and biochemical effects on photosynthesis. In Maple Arrow, where C had the smallest effect on photosynthesis, prolonged CD caused less inhibition of photosynthesis compared to D. Compared to Fiskeby V, the photosynthetic apparatus of Maple Arrow appears to possess superior dark chilling tolerance, a property which probably also conveyed enhanced protection against CD. Proline accumulation was prevented by CD at the ψ_PD where D already resulted in considerable accumulation. The superior capacity for proline accumulation in Maple Arrow would seem to be an important factor in its stress tolerance. Antioxidant activity evoked by CD and D was higher than for C alone. In Fiskeby V, the small increase in ascorbate peroxidase (EC 1.11.1.7) activity, which was in most cases not accompanied by increased gluthatione reductase (EC 1.6.4.2) activity, could impact negatively on its stress tolerance. These results demonstrate large genotypic differences in response to chilling and drought stress, even between soybean cultivars regarded as chilling tolerant.     

Identification and Expression Analysis of Cytokinin Metabolic Genes in Soybean under Normal and Drought Conditions in Relation to Cytokinin Levels

Cytokinins (CKs) mediate cellular responses to drought stress and targeted control of CK metabolism can be used to develop drought-tolerant plants. Aiming to manipulate CK levels to improve drought tolerance of soybean cultivars through genetic engineering of CK metabolic genes, we surveyed the soybean genome and identified 14 CK biosynthetic (isopentenyltransferase, GmIPT) and 17 CK degradative (CK dehydrogenase, GmCKX) genes. Comparative analyses of GmIPTs and GmCKXs with Arabidopsis counterparts revealed their similar architecture. The average numbers of abiotic stress-inducible cis-elements per promoter were 0.4 and 1.2 for GmIPT and GmCKX genes, respectively, suggesting that upregulation of GmCKXs, thereby reduction of CK levels, maybe the major events under abiotic stresses. Indeed, the expression of 12 GmCKX genes was upregulated by dehydration in R2 roots. Overall, the expressions of soybean CK metabolic genes in various tissues at various stages were highly responsive to drought. CK contents in various organs at the reproductive (R2) stage were also determined under well-watered and drought stress conditions. Although tRNA-type GmIPT genes were highly expressed in soybean, cis-zeatin and its derivatives were found at low concentrations. Moreover, reduction of total CK content in R2 leaves under drought was attributable to the decrease in dihydrozeatin levels, suggesting a role of this molecule in regulating soybean's responses to drought stress. Our systematic analysis of the GmIPT and GmCKX families has provided an insight into CK metabolism in soybean under drought stress and a solid foundation for in-depth characterization and future development of improved drought-tolerant soybean cultivars by manipulation of CK levels via biotechnological approach.     

Mycorrhizal impact on drought stress tolerance of rose plants probed by chlorophyll a fluorescence, proline content and visual scoring

Micropropagated rose plants (Rosa hybrida L., cv. New Dawn) were inoculated with the arbuscular mycorrhizal (AM) fungus Glomus intraradices (Schenk and Smith) and subjected to different drought regimens. The dual objectives of these experiments were to investigate the mechanism and the extent to which AM can prevent drought damages and whether physiological analyses reveal enhanced drought tolerance of an economically important plant such as the rose. In a long-term drought experiment with four different water regimens, visual scoring of wilt symptoms affirmed that AM in a selected host–symbiont combination increased plant performance. This effect was mostly expressed if moderate drought stress was constantly applied over a long period. In a short-term experiment in which severe drought stress was implemented and plants were allowed to recover after 4 or 9 days, no visual differences between mycorrhizal and non-mycorrhizal roses were observed. Therefore, the early physiological steps conferring drought tolerance were prone to investigation. Proline content in leaves proved to be an unsuitable marker for AM-induced drought tolerance, whereas analysis of chlorophyll a fluorescence using the JIP test (collecting stress-induced changes of the polyphasic O-J-I-P fluorescence kinetics in a non-destructive tissue screening) was more explanatory. Parameters derived from this test could describe the extent of foliar stress response and help to differentiate physiological mechanisms of stress tolerance. AM led to a more intense electron flow and a higher productive photosynthetic activity at several sites of the photosynthetic electron transport chain. A K step, known as a stress indicator of general character, appeared in the fluorescence transient only in drought-stressed non-mycorrhizal plants; conversely, the data elucidate a stabilising effect of AM on the oxygen-evolving complex at the donor site of photosystem (PS) II and at the electron-transport chain between PS II and PS I. If drought stress intensity was reduced by a prolonged and milder drying phase, these significant tolerance features were less pronounced or missing, indicating a possible threshold level for mycorrhizal tolerance induction.     

Physiological regulation of seed soaking with soybean isoflavones on drought tolerance of Glycine max and Glycine soja

In this study, the Glycine max Jackson cultivar (the relatively drought-sensitive) and the Glycine soja BB52 accession (the drought-tolerant one) were used as the experimental materials. Effects of seed soaking with exogenous soybean isoflavones (daidzin or genistin) on seed germination, and seedlings photosynthesis, relative electrolytic leakage, content of thiobarbituric acid reactive substances , and anti-oxidative activities were investigated under drought stress conditions. The results showed that, treatments of seed soaking with daidzin or genistin could improve seed germination, and alleviate cell damage, enhance anti-oxidative activities and photosynthesis in drought-stressed seedlings, therefore displayed mitigated effects on soybean drought injury, especially for the drought-sensitive G. max Jackson cultivar. Thus seed soaking with exogenous soybean isoflavones may be a usable approach to enhance drought tolerance of cultivated soybean in practice.     

氮素营养与水分胁迫对大豆产量补偿效应的影响

在大豆营养生长期,对大豆进行不同程度的干旱锻炼,同时改变土壤中的施氮水平,研究大豆产量及其构成因子对干旱胁迫复水的反应机制,为大豆节水增产及抗旱机制的实践探索提供理论依据。水分胁迫强度、历时和氮素营养都对大豆产量及其构成因子的补偿效应产生明显影响,水分胁迫抑制了大豆单株粒数的增长,但可以显著提高百粒重;氮素营养会抑制大豆百粒重的增加,但在一定水分条件下可以显著提高单株籽粒的数量,然而随着水分胁迫程度的加重,单株粒数的增加幅度也会相应减少。虽然氮素营养和水分胁迫使大豆产量构成因子产生补偿效应的阈值范围不同,但二者具有一定的耦合区域,在耦合区域内(水分胁迫时间14d左右、土壤含水量为田间持水量的50%—55%、施氮量在97.5—225kg/hm2之间)单株粒数和百粒重都产生较强的补偿效应,二者的协同作用显著提高了大豆的经济产量,使大豆产量表现出较强的补偿效应。结果表明:氮素营养和适度水分胁迫可以通过不同途径提高大豆植株的生长能力,当二者结合后大豆的补偿生长机制更为复杂,最终表现为水分胁迫提高了大豆的百粒重,而氮素增加了大豆单株粒数,二者协同作用使大豆经济产量显著增加。     

Tolerance of Bradyrhizobium japonicum E109 to Osmotic Stress and the Stability of Liquid Inoculants Depend on Growth Phase

Salinity and drought induce osmotic stress in plants and nodulating bacteria. The introduction of soybean in areas with higher soil salt contents or periods of drought pose a challenge for the rhizobial inoculants used to improve nodulation and enhance nitrogen fixation. Bradyrhizobium japonicum is a slow-growing rhizobium used for soybean inoculation that was previously regarded as salt-sensitive. We tested the survival ability of cultures of B. japonicum E109 at the exponential and stationary phases of growth in liquid culture medium against different concentrations of NaCl. We found that stationary-phase cells could tolerate higher levels of salt than exponential-phase cells. This result suggested that the physiological manipulation of the cultures could improve the salt tolerance of this strain. Nonetheless, we also found that exponential-phase cells adapted significantly better to two key situations that a commercial product must face, survival in liquid formulations and survival in soil microcosms resembling conditions of drought. These results suggest that the use of actively growing cells could be an improvement in the production of inoculants. However, it is not cost-effective, because bacteria should be harvested at a time when cell density is lower than that of early stationary-phase cultures, which are normally used in the industry. To overcome this drawback we proved that a fed-batch system can produce exponential-phase cultures with higher cell densities and able to produce liquid inoculants with acceptable survival rates.     

GmPOI gene encoding a Pollen_Ole_e_I conserved domain is involved in response of soybean to various stresses

In the previous research, a novel gene GmPOI (GenBank acc. No. HM235775) encoding a Pollen_Ole_e_I conserved domain was identified in roots of soybean drought resistant cv. Jindou 23. In the present study, GmPOI was cloned and functionally characterized. Real-time quantitative PCR indicated that the expression of GmPOI was induced by drought, cold, salt and abscisic acid in wild-type soybean. The soybean plants overexpressing GmPOI showed higher tolerance to drought stress than wild types. We concluded that GmPOI is probably a novel gene that is involved in the response to various stresses in soybean.     

干旱胁迫对苗期大豆异黄酮合成的影响

以不同耐旱性的2个大豆品种(高耐旱JP-6、低耐旱JP-16)为研究材料,采用高效液相色谱和实时荧光定量PCR技术,分析不同时间持续干旱胁迫下,大豆叶片和根系中异黄酮的积累变化及关键酶基因的表达情况.结果表明:大豆根部异黄酮含量显著高于叶部,而异黄酮关键酶基因的表达量则在叶片中更高,耐旱品种JP-6根部的异黄酮积累量更大.随着干旱胁迫持续时间的增加,不同耐旱品种的异黄酮合成与积累变化规律存在显著差异:强耐旱品种JP-6的根和叶中,异黄酮积累量均呈现先下降后升高的趋势;而弱耐旱品种JP-16则相反,异黄酮积累量在不同部位中均呈现先上升后降低的趋势;除JP-6叶中C4H、4CL、IFS2等异黄酮合成上游基因外,其他不同品种、不同部位的关键酶基因表达量均随着干旱胁迫持续时间的增加,呈现先下降后上升的趋势.大豆叶片是异黄酮的主要合成部位,大豆根部也存在少量的异黄酮合成.弱耐旱大豆根部的异黄酮合成和最终积累量均较低,强耐旱品种则较高.根部异黄酮积累量高的大豆品种,其耐旱性更强.     

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.