Medicinal plants are used to treat several illnesses linked to oxidative stress. The herbaceous annual plant Heliotropium indicum L. is found in Asia and is widely used in traditional medicine. The investigation aimed to evaluate the antioxidant prop-erties of five solvent extracts (hexane, chloroform, ethyl acetate, methanol, and aqueous) of Heliotropium indicum L. roots. The antioxidant compounds in the ethyl acetate extract were also identified. The ethyl acetate root extract demonstrated significant antioxidant properties in DPPH radical scavenging and reducing power assays. Shimadzu-QP2020 GC-MS was used to assess the phytochemical composition of the ethyl acetate root extract. The extract contained chemicals whose mass spectra matched those in the National Institute of Standards and Technology (NIST) library. Conventionally, the combined analytical method of gas chromatogra-phy-mass spectroscopy (GC-MS) is employed to identify and ascertain the substances in the sample. The functional groups in the ethyl acetate extract were analyzed by FTIR spectroscopy. The GC-MS analysis of ethyl acetate root extract identified forty-two compounds in total, with the highest area percentage (23.11%) being Cholesta-22,24-dien-5-ol, 4,4-dimethyl-and the lowest area percentage (0.26%) being 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione. The GC-MS investigation revealed twelve major compounds. This work is the first to report on the antioxidant, statistical analy-sis using ANOVA and Post-hoc Tukey's HSD test (SPSS 27.0 software), GC-MS, and FTIR analyses of Heliotropium indicum L. ethyl acetate root extract. The current study offers a foundation for natural chemicals that are safer and less expensive to combat illnesses linked to oxidative stress.
Studying the growth of soybean roots in hydroponic culture provides a means to avoid issues caused by the heterogeneity and opaqueness of soil. In this study, we applied three salt-tolerant soybean near isogenic lines (NILs) and three corresponding salt-sensitive NILs for hydroculture with ten different salt concentrations. A digital camera was utilized to continuously capture RGB side-view images of hydroponically grown soybean roots. To extract two-dimensional information of root area from the side-view images, we applied a machine learning-based method, the trainable WEKA segmentation. The root volume was estimated by raising the root area to the power of 3/2 and applying a coefficient. Additionally, skeleton length of root was obtained from the bottom 30% of images with minimal root overlap to calculate the average root diameter. The total root length was estimated from the root volume and the average root diameter then applying a coefficient. The results indicate that the coefficients of root volume and total root length ranged from 0.0164 to 0.0291 and 0.0449 to 0.0874, respectively, for the three correspondent NILs. Using these coefficients, the root volume and total root length were estimated with the root mean square error (RMSE) ranging from 1.87 to 5.32 cm3 and 0.67 to 2.32 x 103 cm, respectively. The estimation results clearly demonstrated the differences across NILs and treatments even though ten salt treatments caused different root morphologies. This method may contribute to further understanding the dynamic changes in root traits under various abiotic stresses.
Drought limits water availability and negatively impacts rice growth. One of the strategies to overcome drought stress is the genetic improvement of root traits by marker-assisted selection (MAS). Using the MAS strategy, pyramiding quantitative trait loci (QTLs) efficiently stacks multiple QTLs. To elucidate the effects of pyramiding root QTLs on shoot production under drought, we used near-isogenic lines (NILs) and pyramided lines (PYLs), introducing single or multiple QTLs derived from Kinandang Patong (upland rice) into IR64 (lowland rice). We selected one QTL from each root morphological trait: root growth angle (DRO1), root thickness (Sta1), root length (qRL6.1), and root volume (qFSR4). We evaluated the effects of pyramiding root QTLs on root system architecture (RSA). In the basket method and 3D imaging by X-ray computed tomography, all NIL and PYLs with DRO1 showed robustly steeper root growth angle. In a hydroponic culture assay, root length QTL qRL6. 1 increased maximum root length. In a lysimeter experiment, root volume QTL qFSR4 increased root volume per stem. Next, we examined the NILs and PYLs for shoot biomass production under drought conditions in lysimeter and field experiments. Two lines (DRO1-NIL and Sta1+DRO1-PYL) had a higher shoot biomass production under drought than IR64. DRO1-NIL exhibited deeper RSAs and increased water uptake compared to IR64. Sta1+DRO1-PYL was not as deep as DRO1-NIL but had a higher normalized difference vegetation index, lower canopy temperature, and higher shoot biomass in the field experiment. In summary, DRO 1 plays a critical role in RSA, and pyramiding Sta1 with DRO1 increases shoot biomass during drought. This suggests the pyramiding of root QTLs could improve RSA and enhance biomass production under drought stress.
Root system traits are important for crop production. This study characterized the soybean root system by analyzing position markers and digital images. Soybean genotypes were grown in a growth pouch, and their root systems were photographed every few days using a digital camera. Image analysis was performed to segment the roots and measure root length. Three genotypes widely used to provide reference genomes or as major breeding materials were used for Experiment 1. The root dry weight of 'Williams 82' at 13 days after transplant (DAT) did not differ significantly with that of 'Enrei'. However, the root length of 'Williams 82' at 8-11 DAT was significantly smaller, whereas that at 13 DAT was comparable with that of 'Enrei'. Determining the differences by sampling date and root segment revealed that 'Williams 82' developed its root system evenly from the middle and bottom segments at 8 DAT, whereas those of 'Enrei' and 'Peking' developed unevenly from only the bottom segment. Because the genotypes for Experiment 1 had different seed sizes and extent of root elongation, the genotypes with similar seed sizes were used for Experiment 2. Compared with 'U 1042-1', 'COL/PAK/1989/IBPGR/2326(1)' showed more elongation in the middle root segment at 8-11 DAT. Image analysis enabled a more in-depth investigation of root system development over time and by root segment.
Soybean ( Glycine max L.) can establish a symbiotic relationship with rhizobia. However, little is known about the host plant genes that correlate with the nitrogen fixation activity of symbiotic rhizobia. In this study, we investigated soybean genes that correlate with bacterial symbiotic nitrogen fixation in two experiments. In the first experiment, soybean seeds were inoculated with two strains of soybean rhizobia exhibiting different nitrogen fixation activities and were grown in the field until the R1 stage. Subsequently, the roots and nodules were subjected to RNA-seq analysis to identify the expression of soybean genes related to bacterial nitrogen fixation. In the second experiment, to confirm host gene expressions dependent on bacterial nitrogen fixation activity, nod+/fix-(Delta nif gene) mutants and the wild type were inoculated into soybean, and gene expressions were evaluated by qRT-PCR. GmNRT2.5, a high-affinity nitrate transporter gene, was correlated with nitrogen fixation activity. qPCR analysis of roots and nodules inoculated with a nod+/fix-mutant revealed that GmNRT2.5 on chromosome 8 (Glyma.08G284000: GmNRT2.5 Chr8) was particularly correlated with nitrogen fixation activity in the root nodule section. Based on the results, it is suggested that GmNRT2.5 Chr8 may function as a regulatory gene bridging nitrogen fixation and nitrate absorption from the soil. Therefore, GmNRT2.5 Chr8 might be a useful host gene for estimating symbiotic nitrogen fixation activity.
Knowledge of biodiversity of soil microorganisms, including arbuscular mycorrhizal fungal (AMF) species, is limited in agroecological contexts. We investigated the effects of the popular inoculant Dr. Kinkon R10 on AMF communities in rice and pearl millet in upland and lowland fields under different water regimes by using a metagenomic approach. Numbers of operational taxonomic units (OTUs) were much lower in lowland than in upland. In upland, alpha diversity was lowest in rice but highest in pearl millet under the nonirrigated treatment (W0); OTUs of Claroideoglomus showed the greatest differences in abundance. Three OTUs of Claroideoglomus tended to have higher abundance in pearl millet under W0, whereas that of two OTUs ( Acaulospora , Diversispora) ) increased under the well-irrigated treatment (W100). In lowland, alpha diversity was higher under the flooded condition than alternate wetting and drying, with greater abundance of Acaulospora, , Ambispora, , and Scutellospora. . Overall, inoculation with R10 did not change the alpha and beta diversity of AMF communities. In upland, eight OTUs showed greater abundance in pearl millet than in rice, including one OTU present in the R10 inoculant. In upland, the shallow soil layer (0-10 cm) had greater abundance of Claroideoglomus, , Paraglomus, , and Acaulospora, , with a higher diversity index, whereas the deep layer (20-30 cm) had greater abundance of Rhizophagus and Scutellospora. . This study highlighted abundance of Acaulospora in higher water regimes in both upland and lowland and of Claroideoglomus for pearl millet in lower water regimes in upland.
Investigating effects of introgressed root- trait QTLs in field environments is a challenging task. Seven rice BC3F4 3 F 4 lines with at least one of six QTLs for root vertical distribution ( DRO1 , DRO2, , DRO3, , qFSR4, , qRL6.1, , QRO2) ) introgressed into a popular Colombian variety, FEDEARROZ 60, were evaluated for changes in root traits together with a BC3F4 3 F 4 line lacking root QTLs and the parent FEDEARROZ 60. A flooded experiment with deep-flooded and shallow-flooded treatments and an aerobic experiment using a raised-bed system were repeated in two seasons in Central Colombia. In the flooded experiment, ST604_302, containing DRO2, , showed the highest root weight and root weight proportion in the 20-30-cm soil layer. The average root weight and proportion of roots at the 10-20-cm soil depth and average root weight at the 20-30-cm depth were significantly greater for the lines containing DRO2 than for those without. In the aerobic experiment, the four lines with DRO2 had higher deep-root ratios than the other genotypes. In the aerobic experiment, a higher deep-root ratio did not lead to greater root weight at either depth examined; the root weight parameters showed genotype x season interaction, and genotype had no significant effect on root weight. In the flooded experiment, the four DRO2-introgressed lines showed higher root length density below 10-cm depth than other lines in the deep-flooded treatment but not in the shallow-flooded treatment. These results showed limited but positive changes in deep-root growth in field environments through root-QTL introgression, and revealed interactions with water management and season.
The influence of training methods on the yields of cucumber (Cucumis sativus) was investigated in the aspects of root characteristics. The experiment was conducted in the new Nutrient Film Technique hydroponics system specially developed for cucumber production (Mitsubishi Chemical Aqua Solutions, Co., Ltd., Tokyo, Japan).The lowering training (LT) and the pinching training (PT) were used to manage the cucumber plant's canopy during the growth cycle. At the end of the experiment, the xylem sap bleeding rate, the root biomass, the remaining aerial biomass and the main stem diameter were measured. In addition, individual fresh and dry weights of matured leaves at the lower, middle and upper parts of the plant's canopy were measured during the growth period. The recorded fruits yield was used for a correlation test with the xylem sap bleeding rate. The results indicated that the sap bleeding rate, root dry weight and root dry and fresh weights ratio were significantly higher in LT treatment. Similarly, the remaining aerial biomass fresh weight was significantly greater in the same treatment. On the other hand, individual leaf fresh and dry weights and the main stem diameter were significantly greater in the PT treatment, at the middle and upper parts of the plant's canopy. These results were explained by the fact that in the PT treatment, more carbohydrates produced by plants were apparently used to re-establish the pinched shoots at the expense of root production. Moreover, after pinching the lateral shoots, biomass production was improved due to the penetration of sunlight in the plant's canopy. Considering the relationships between root traits and plant productivity, the LT method is suitable for a long-term cultivation due to its faster sap bleeding rate, which is necessary to stimulate root physiological activity and promote plant biomass production.
The effects of hydrogen sulfide (H2S), re-leased from the donor sodium hydrosulfide (NaHS), on maize seedlings grown hydroponically for 6 days were investigated. Plant biomass, malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide (O2 center dot-) content, and root exudates (organic acids) were measured. Results showed that 100 and 200 pM NaHS is the most appropriate and suitable concen-tration for the growth and development of maize seedlings, without affecting the MDA and H2O2con-tents but altering the O2 center dot-. In addition, high concen-trations of 500 and 1000 pM NaHS adversely af-fected these parameters compared with the control (CK). The pH of the root exudates declined under NaHS treatments. The organic acids in the root ex-udates, including fumaric, acetic, formic, and malic acids exhibited higher contents at 100 pM NaHS treatment, the lactic and citric acids were higher at both 100 and 200 pM NaHS. In contrast, oxalic acid was reduced at all NaHS concentrations compared with the CK. Low contents of all the organic acids analyzed were found under 500 and 1000 pM NaHS treatment. In conclusion, all the above pa-rameters were affected by the application of NaHS, while higher NaHS concentration was toxic for maize seedlings.
The root hydraulic conductance of rice is often measured using the pressure chamber meth-od only for the main stem at the seedling stage, as it is difficult to evaluate at a more advanced growth stage with tillers due to the high risk of pressure leakage from the gaps between the tillers. The aim of this study was to identify techniques that are ef-fective for prevention of air leakage and an im-provement in the success rate of root hydraulic conductance measurements in rice plants with til-lers. Using three rice (Oryza sativa L.) genotypes, FR13A (aus), KDML105 (indica), and Swarna (indi-ca), the root hydraulic conductance of the main stem and four tillers were calculated using the pres-sure chamber method at 98, 104, and 95 days after sowing, respectively, using the following three tech-niques: 1) careful detachment of dry leaf sheaths, 2) ensuring spacing between tillers and an appro-priate amount of silicone in the socket, and 3) pre-fixing the socket to prevent vertical misalignment. Using these three techniques, we achieved a suc-cess rate of 92.8% (13 of 14 plants) for root hy-draulic conductance measurements in the geno-types. Additionally, we show that genotypic variation in root hydraulic conductance exists at the late vegetative stage, and that the growth stage can have a significant effect on root hydraulic con-ductance values. In conclusion, this study presents a detailed pressure chamber method for measuring root hydraulic conductance in rice plants with tillers, reducing the risk of pressure leakage, and improv-ing the success rate of the measurement.
Sugarcane (Saccharum spp.) growth and yield decrease in acidic soil (low pH and high Al con-tent) conditions owing to impaired nutrient and water absorption. An efficient method to evaluate acid tol-erance needs to be developed to utilize Erianthus arundinaceus to improve acid tolerance in sugar-cane. Herein, we performed a root electrolyte leak-age assay to quantitatively evaluate and compare the acid tolerance of the roots of sugarcane cultivar NiF8 and E. arundinaceus accession JW630 under various treatments of acid solutions in laboratory. Additionally, cellular damage of root was observed anatomically and their growth in acidic soil was veri-fied by pot cultivation. To detect the interspecific dif-ference in injury index of root by measuring electro-lyte leakage, the following method was found suitable: initial washing for 15 min, acid stress treat-ment with 3% Al2(SO4)3 solution for 2 h, and leaking electrolyte from the damaged cells in ultrapure water for 2-4 h. The index was aligned with the relative growth rate (RGR) under acidic soil (pH 5.0), as E. arundinaceus with a lower index value exhibited a higher RGR. Callose, indicative of cellular damage, was deposited both after exposure to short-term and long-term acid stress in the exodermis of sugarcane, which showed higher injury index and lower RGR than those of E. arundinaceus, whereas callose de-position was not observed in E. arundinaceus even after acid stress treatments. Our results strongly suggest that the injury index measurements can be used to quantitatively evaluate differences in acid tolerance between sugarcane and Erianthus roots.
In this study, two experiments were conducted to evaluate the genotypic variation of rice root system distribution and root activity in response to short-term drought conditions. Seven rice genotypes were used, of which one (Rexmont) showed the greatest reduction in shoot biomass under drought, and two (Swarna and KDML105) showed the least reduction in shoot biomass under drought in both experiments. In a phytotron experiment (Experiment 1) in which root hydraulic conductivity (Lpr) of 21-day-old rice plants was evaluated in well-watered (control) and dry down (drought) conditions, the Lpr of Swarna, KDML105, and IRAT109 were significantly lower under drought compared to the control. In a field experiment (Experiment 2) conducted in the 2013 wet season at IRRI, stomatal conductance, bleeding rate, and root surface area density (RSAD) at 0-15, 15-30, 30-45, and 45-60 cm soil depths were measured in an irrigated (control) and rainfed (drought) treatments. Swarna, KDML105, and FR13A showed significant reductions in RSAD at 0-30 cm depth under drought in the field compared to the control, while Rexmont and IRAT109 showed no significant changes. In addition, Rexmont and Swarna both maintained higher bleeding rates than the other genotypes. Based on the root hydraulic and architectural traits of contrasting genotypes, we conclude that the bleeding rate did not explain the genotypic variations in the maintenance of shoot biomass, and that reducing shallow root growth and Lpr in response to drought conferred the best ability to maintain shoot biomass under short-term drought conditions.
SOil-surf ace Roots (SORs) are roots that elongate over or near the soil surface. They allow plants to escape waterlogging-caused hy-poxia and high salinity that are often present in deeper soil. Quantitative evaluation is essential for understanding SOR function and identifying the genes and quantitative trait loci (QTLs) that control it. However, existing methods to evaluate SOR for-mation are laborious/slow or semiquantitative. Here, we describe a high-throughput quantitative SOR evaluation system that uses an overhead scanner. We evaluated SORs in 56 wild rice (Ory-za glumaepatula) introgression lines (ILs) with the genetic background of O. sativa. The method can quantify the SOR area of a plant in less than 90 sec. During the image analysis process, multi -threshold segmentation on the scanned images re-duced the reflection noise of the digital image and improved the accuracy of the estimation of SORs formation. The root surface areas of SORs of the O. glumaepatula ILs varied widely, which should make them well-suited for identifying SOR-related QTLs. The overhead scanner method has the po-tential to quantitatively and rapidly evaluate SORs developed on the soil surface.
Strontium (Sr) is a known non-essential element for plants. However, its toxic effects at high concentration on plants remain unclear. Here the effects of Sr on the growth and phytotoxicity were investigated in Arabidopsis thaliana. The plants grown on a medium containing 10-mM Sr showed reduced root fresh weight (FW) and root tration could inhibit root growth. Assessment of Sr in the shoots and roots indicated that its concentradose-dependent manner, while photosynthetic pigconfirmed that cell death was induced by ROS.
Exploring new plant-associated rhizo-bacteria and reintroducing them to the crop-soil en-vironment is among the strategies to reverse the declining quality of the agricultural soil environment. A total of 340 isolates were recovered from the rhi-zospheres of okra (Abelmoschus esculentus), leaf mustard (Brassica juncea), and brinjal (Solanum melongena). From among the total, 155 isolates (45.59%) were diazotrophs, and 122 (35.88%) and 127 (37.35%) were isolates with the solubilising ac-tivity of phosphate and potassium, respectively. Thirteen of the most promising isolates were identi-fied by gas chromatography for their cellular fatty acid methyl esters: Escherichia vulneris (TC19), Klebsiella pneumoniae (BA5, SA19, SB22), Pan-toea ananatis (TC22), Pseudomonas aeruginosa (BA46), Pseudomonas putida (BA29, BA37, SC5, SC14), Salmonella bongori (BC17), Salmonella en-terica (BB2), and Shigella dysenteriae (BA16). Three Pseudomonas strains viz. P. aeruginosa BA46 and P. putida BA37 and SC5 were selected after enhancing root elongation and vigour of let-tuce seedlings. The American lettuce growth per-formance in the non-circulating hydroponic system was established. The inoculation with rhizobacteria strains, SC5 and BA46, stimulated shoot and root biomass over uninoculated control plants. The in-oculation of lettuce plants with isolates BA37, BA46 and SC5, produced significantly longer roots com-pared to uninoculated control plants. This study in-dicates the potential use of the strains BA46 and SC5 as candidates for the formulation and produc-tion of hydroponic nutrient solution fortified with beneficial bacteria.
The root growth angle (RGA) is an im-portant breeding target that confers high crop adapt-ability to deleterious environments. In barley, natural variations in RGA among accessions have been ob-served, but many of the genetic factors that cause of this variation remains unclear. In this study, we explored the orthologs of OsDRO1 (DEEPER ROOTING 1) and OsqSOR1 (quantitative trait lo-cus for SOIL SURFACE ROOTING 1), which play a critical role in RGA regulation in rice, from barley genome and analyzed the polymorphisms of these genes among barley accessions. BLASTP search detected putative orthologs of OsDRO1 and Osq-SOR1 in barley (HvDRO1 and HvqSOR1) with more than 60% amino acid similarity. Sequence analysis identified SNPs causing mis-splicing and nonsynonymous amino acid substitution in HvDRO1 and HvqSOR1, respectively. These SNPs were associated with RGA variation among the 47 barley accessions. Phylogenetic analysis using the 105 barley accessions revealed that the alleles of HvDRO1 and HvqSOR1 are related to the genetic background of the accessions. Further-more, the mutant allele of HvDRO1 is mainly shared in the Hokuriku/Nagano subpopulation, suggesting that the mutant allele is involved in lo-cal adaptation of barley cultivars to the soil envi-ronment of the region. Our findings suggest that the polymorphisms of HvDRO1 and HvqSOR1 are possible determinants of RGA variation in barley, at least in Japanese accessions, and provide infor-mation on allelic variants of the genes for marker -assisted selection to genetic improvement of RGA of barley.
A rice root system consists of main roots, and lateral roots which account for a large portion of the total length and surface area of the whole root system, indicating that lateral roots greatly contribute to its hydraulic conductance. It is therefore worthwhile measuring aquaporin activi-ties of lateral roots in comparison with main roots to evaluate their contribution to hydraulic conduc-tivity. However, the sampling of fine lateral roots is time-consuming, and its method can be critical be-cause of the difficulties of handling them for sam-pling. In addition, root has been reported to be sensitive to the surrounding environment and af-fected by destructive sampling procedures in which lateral roots are removed from main roots. There-fore, in this study, we attempted to establish a method for collecting samples of lateral and main roots for aquaporin analysis while minimizing the effect of separation of roots from the shoot, and the removable of lateral roots from main roots on the transcript abundance. We conducted three ex-periments and measured the expression levels of four aquaporin genes including OsPIP2;1, Os-PIP2;4, OsPIP2;5 and OsTIP2;1 with different sampling time length (experiment 1), with or with-out shoot removal (experiment 2) and light and dark periods samplings with different light condi-tions (experiment 3). Our results revealed that the aquaporin expression levels in separated roots did not change compared to those of the roots which were immediately preserved after sampling from the shoot, when root separation was conducted within 10 minutes without shoot removal under flu-orescent light during light period of growth, as well as under indirect lighting during dark period of growth.
