Peanut root invasion by <i> Bradyrhizobia </i> is through a crack entry, which is different from many other legumes applying an infection thread entry in root hair. Understanding the role of root hair in the crack entry of <i> Bradyrhizobia </i> invasion of peanut root and subsequent peanut nodulation would facilitate improvement of biological nitrogen fixation in cultivated peanut. The objective of this study was to investigate the involvement of root hair in <i> Bradyrhizobial </i> invasion of peanut. Seedling roots of a nodulating peanut cultivar were observed for root hair emergence, its life span, and nodule formation at the base of the lateral roots with and without rhizobia inoculation for 14 days after germination (DAG). Scanning electron microscopy (SEM) was utilized to observe rhizobia accumulation at lateral roots at 24 hours after inoculation (HAI) before the emergence of root hair. Root hair emerged at 7 DAG with or without rhizobia inoculation. Two variations of rosette hair (RoH) were observed, the transient-thin RoH had life span of 3 days after root hair emergence and the thick and densely distributed RoH type stayed till the time of nodule emergence (9 days after inoculation). The lateral root devoid of root hair at the top 2 cm region was found to produce nodules. The SEM observation of seedling roots at 24 HAI showed that <i> Bradyrhizobia </i> invaded the roots at epidermis, protoplasm of cortical cell, and cortical cells of the main root near the newly emerged lateral root in the absence of RoH. The observations validated that root hair is not required in the <i> Bradyrhizobia </i> invasion of peanut root in the crack entry mode. Results from this study provided important morphological information for the hypothesis of close relationship between RoH and peanut nodulation for further genetic study of crack entry mechanism and signaling pathway of symbiosis between <i> Bradyrhizobia </i> and peanut .
Legumes can form a symbiotic relationship with rhizobia to fix nitrogen in root nodules, where the rhizobia reduce atmospheric nitrogen into ammonia (a process known as biological nitrogen fixation-BNF) to supply the hosts’ nitrogen need and in return to get nutrients from the host plant. For the symbiotic association, rhizobia must firstly interact with and enter the host root cells. The root epidermis, as a barrier, must be overridden by rhizobia for infection [
The most characterized infection path of rhizobia into host roots is through the intracellular infection thread (IT), which is employed by approximately 75% of legume species including temperate and tropical/subtropical legumes such as Vicia, Pisum Glycine, and Phaseolus [
A less studied and alternative mode of infection is intercellular crack entry, which is employed by about 25% of the legume species [
Cultivated peanut (Arachis hypogaea L.) is a legume crop of immense agricultural and economic importance with numerous uses in several food products. It belongs to the Fabaceae family, sub-family Papiliononoideae [
Several studies indicated that for intercellular crack entry, rhizobia invade the root at the site of lateral root emergence but none of these studies addressed the role of RoH [
The specific relationship between root hair and peanut nodulation is puzzled. Is RoH needed for Bradyrhizobia entry into the root cells of peanut for nodulation process to happen or the genes controlling nodulation in peanut also control RoH development? To provide an initial answer to this question, we investigated the involvement of peanut RoH in peanut root invasion by Bradyrhizobial and nodulation. Outcome from this study will clarify whether root hair found at the base of lateral roots are required for Bradyrhizobial invasion process of cultivated peanut root, and the association of RoH with nodulation of cultivated peanut. The results will bridge knowledge gaps of genetic and physiological basis of peanut nodulation and contribute to future studies of peanut symbiosis for BNF improvement in peanut and other legume or non-legume species.
Seeds of Tifrunner, a normal nodulating peanut cultivar [
Bradyrhizobia strain LB8 [
At 5 DAG, roots of individual peanut seedlings of both inoculated and non-inoculated groups were observed for appearance of root hair daily for two weeks. Lamp Black (Natural Pigment LLC) water solution was used to mark the secondary roots emerged on 5 DAG or on the day of inoculation or on 0 day after inoculation (DAI). Pictures of peanut roots were taken daily for two weeks using an Olympus MVX-TV1X Cdigital camera and iPAD.
The seedlings of nodulating cultivar Tifrunner were inoculated with Bradyrhizobia strain LB8 at 5 DAG. The top 2 cm root segments close to the hypocotyl region, where not every base of lateral root had RoH, were observed for root hair and nodule development for two weeks to investigate if the presence of root hair is required for nodulation in these top 2 cm root segments. Pictures were taken daily until 16 DAI (i.e. 21 DAG) using an Olympus MVX-TV1XC digital camera and presence and absence of nodule were recorded.
Scanning electron microscopy was performed on peanut root as described by Walter et al. [
Three regions (0.5 - 1 cm, 1 cm - 2 cm and 2 cm above) of the roots were observed for the occurrence of root hair on their lateral roots for three seedlings (or three replicates). The data were analyzed using analysis of variance (ANOVA) to determine the RoH distribution in different segments of the tap root in Excel. The least significant difference (LSD) test was performed to determine the statistically significant differences of distribution of RoH in the tap root segments. A p value < 0.05 was considered as the threshold for statistical significance between different data points.
Roots of individual peanut seedling of both inoculated and non-inoculated were observed daily for the appearance of RoH and the life span of the appeared RoH were monitored daily till they disappeared or senesced. Our observation showed that RoH started to appear at 7 DAG of both inoculated and un-inoculated peanut roots at the base of the lateral roots when the main roots were approximately 6 cm for both transient-thin root hair and densely populated RoH (
Among the observed roots of nodulating plants, we noticed that not every single lateral root had RoHat their base especially at the upper part (0.5 cm - 2 cm) of the tap root close to the hypocotyl region (
Peanut root at 5 DAG, before the emergence of RoH, was inoculated with Bradyrhizobia strain LB8. At 24 hours after inoculation (HAI), when no RoH (normally appear at 7DAG) was observed yet (
The natural capability of legumes to mutualistically associate with rhizobia to convert atmospheric nitrogen into nitrogen nutrition for plants plays a critical role in maintaining the sustainability of agriculture. The notion that different entry routes exist for rhizobia to invade the host and to form functional nodules has attracted the attention of researchers [
mechanism than the IT mode of invasion [
We found that RoH started to appear at the base of lateral roots at 7 DAG with or without rhizobia infection, indicating that RoH is not induced by Bradyrhizobial inoculation. However, Wissuwa and Ae [
RoH formed. The hairs disappeared at 3 DARHE were transiently-thin root hair while the RoH with extended time of stay were typically thick and dense. We also noticed that the dense RoHs were typically present on actively elongating root regions of the seedlings (from 2 cm region of the tap root downward), while none or few RoHson older root regions (ranging from 0.5 cm - 1 cm top segment of the tap root) during our two-week observation window. This observation is in agreement with previous findings [
Tifrunner, a normal nodulating peanut cultivar, produces nodules along the roots. However, we noticed that not every lateral root had nodules or RoH at its base, which provided us the opportunity to investigate the relationship between RoH and nodule emergence sites. We observed that there was nodule development from a lateral root base devoid of RoH. This is our first evidence to support that RoH is not required for Bradyrhizobial invasion of peanut root and subsequently not required for peanut nodulation, which is in line with previous observation [
Karas et al. [
Based on these observations, we hypothesized that the genes controlling nodulation most likely also have pleiotropic effect on the development or appearance of RoH. Pleiotropic effect of genes is not rare cases and there are quite a few reports on this effect. For example, three mlo powdery mildew resistance genes also have impacts on barley yield [
Documenting the complete basic information of peanut root, root hair, and nodule formation is very important in studying genetic and molecular mechanisms of peanut nodulation. In this study, we observed the dynamics of RoH and its involvement in Bradyrhizobial invasion of cultivated peanut root to investigate its involvement in peanut nodulation. The two weeks observation of cultivated peanut roots at the top segment (0.5 cm - 2 cm) of tap root devoid of RoH, which eventually produced nodules illustrated that RoH is not required for nodulation process and the SEM study corroborated this claim. This conclusion further directs us to the speculation that there are pleiotropic effects of the genetic components controlling peanut nodulation and RoH formation. Since many phytohormones are shared between root hair formation and nodule development in M. truncatula and L. japonicus especially the positive regulation of auxin in both mechanisms, we hypothesized that these two lateral developmental programs may share similar phytohormones in cultivated peanut. The results resolved the unclear involvement of RoH in crack entry invasion process found in cultivated peanut, which could further guide in the quest of identifying the genes controlling peanut nodulation and eventually in plant nitrogen fixation efficiency improvement.
The authors declare no conflict of interest.
This project is funded by Early Career Fund by Institute of Food and Agricultural Sciences (IFAS), University of Florida; Florida Peanut Producers Association; and Tertiary Education Trust Fund (TETFUND) through Adekunle Ajasin University, Nigeria.
Maku, J., Wang, L.P., Liu, F.X., Liu, L.X., Kelley, K., Peng, Z. and Wang, J.P. (2018) Involvement of Root Hair during Rhizobial Invasion in Cultivated Peanut (Arachis hypogaea L.). American Journal of Plant Sciences, 9, 1646-1659. https://doi.org/10.4236/ajps.2018.98119