Two soil experiments were performed. In the first experiment, four cotton plants were grown for four months. For these experiments, the plants were positioned with the root crown approximately 8 cm deep . In the second experiment, a pregerminated maize seed was planted 3 cm deep and then grown for four months .The iCSD results showed that the current leakage occurred in the very proximal regions of the root systems in both soil and hydroponic conditions. The proximal leakage was observed despite the return electrode being placed at the bottom of the rhizotron to allow deep current pathways. Nonetheless, the expected influence of the return electrode position was observed in the laboratory test with metallic roots and motivates the use of this electrode configuration in future laboratory experiments. Our results are consistent with the early studies on maize root electrical properties , and corroborate the recent works that questioned the assumptions of the BIA methods . The high resistivity of the top layer of soil is expected to induce root suberization : our results would support the physiological hypothesis of water and nutrient absorption through older and possibly suberized roots . It is worth noticing that the absence of current leakage along the section of the cotton stems in the top dry soil supports the assumption that the electrical structure of roots controls their current conduction behavior. Suberized epidermal cells can affect the movement of ions and, consequently,ebb and flow bench the current conduction in roots . Therefore, it is feasible for current to be conducted along deeper portions of the more woody roots with minor leakage.
The BIA experimental results that have observed positive correlations between electrical signals and root area are likely a result of physiological correlations between the root regions that contribute to the current flow and hair roots, which contributes most to functional root surface area . While correlations between BIA electric signals and investigated root traits appear to be indirect, the correlations observed across experimental platforms and species continue to validate its value for in-situ root phenotyping . In their study on field grapevines, Mary et al. concluded that the CSD could be used to infer the root depth distribution. Because of the significant suberization of major roots in grapevines and orange trees, the electric current could penetrate deep into the root system before significant leakage occured. The deeper current penetration allowed the iCSD method to access and phenotype the root system. On the contrary, limited current results in lower sensitivity of the iCSD to distal and younger parts of the root system that are likely dominated by less suberized, finer roots. We attribute differences in current penetration among root systems of maize, cotton, grapevine, and orange tree to the differences in physiological traits such as the extent of suberization and lignification. If on one hand the sensitivity to the root physiological traitsis a promising opportunity, on the other hand it has to be accounted for when phenotyping more herbaceous roots.To assess the involvement of the MsLEC1 and MsLEC2 genes in nodulation of alfalfa, we examined the responses of rooted cuttings of transgenic vector control plants, plants expressing the antisense transgene for MsLEC2 , and plants expressing the antisense transgene for MsLEC1to inoculation with -glucuronidase – or green fluorescent protein -marked strains of S. meliloti Rm1021. Because legume lectins have been associated with facilitation of nodulation, reduced nodulation of lectin loss-of function plants was predicted. However, contrary to our expectations, all the transgenic plants, including the controls, were nodulated 7 days post inoculation .
By this time, the LEC1AS plant lines had already developed abnormally large numbers of nodules . The colonized nodules, as evidenced by the presence of GUS- or GFP-marked rhizobia, were frequently adjacent to each other or directly opposite one another on the root. Infection thread development in root hairs, as viewed by fluorescent microscopy of GFP-marked Rm1021, was not impaired in the LEC1AS roots, although some of the nodules appeared to be uninfected . Occasionally, uninfected nodules also developed on the roots of LEC2AS plants , but generally, the LEC2AS root nodules contained the marked strains . With one exception, line 49b, the LEC2AS roots developed markedly separate rather than clustered nodules. By 12 to 14 dpi, many of the LEC1AS nodules were already beginning to show signs of senescence, as indicated by the reduction in overall staining in a nodule 13 dpi with a GUSmarked strain and by the decrease in Rm1021 GFP fluorescence in the center part of a 2-week-old nodule . In the Rm1021 GFP nodules, there was a concomitant accumulation of autofluorescent compounds, presumably flavonoids, in the central and proximal parts of the nodule . Sections of senescent nodules demonstrated that the rhizobia had senesced from the inside outward . In contrast, the vector control and LEC2AS nodules as well as the plant lines containing the cognate sense transgenes did not show any symptoms of senescence until many weeks after inoculation. All nodules were colonized by rhizobia . Nodules formed on vector control plants grown in potting soil appeared identical, i.e. pink and elongated, to nodules formed by nontransgenic, wild type alfalfa . Similarly, nodules formed on the LEC2AS plants were elongated and pink in color . In contrast, although some LEC1AS root nodules appeared morphologically normal, many LEC1AS nodules were large, multilobed structures that showed signs of senescence, including loss of pink color due to breakdown of leghemoglobin, whereas others showed arrested development at early stages of nodule formation .
Nodulation was also examined in an inert root medium . The Turface grown plants exhibited an identical nodulation phenotype . The LEC1AS lines displaying the most severe developmental and reproductive abnormalities had the highest proportion of abnormal nodules. We also examined plants that expressed the sense transgene for MsLEC1 and found that these roots also developed some large, albeit pink, multilobed nodules , suggesting that cosuppression might be the cause. We did not pursue this analysis further. In contrast, the LEC2ST plants produced nodules that were identical to those of the vector control .When grown hydroponically, the differences between inoculated versus uninoculated plants became very obvious. The shoots of the uninoculated plants were paler than the nodulated plants and, of the three types of transgenic plants, the LEC1AS plants were the most chlorotic . The differences between the vector control, LEC2AS, and LEC1AS plants 35 dpi, were striking . The LEC1AS plants were much less robust than either the vector control or the LEC2AS plants; the plants were consistently small and chlorotic, with a poorly developed root system. Nevertheless, a number of large, prominent nodules were observed on the roots of the LEC1AS plants , as well as small, senescent nodules . The abnormal nodulation phenotype in LEC1AS plants was evident 15 to 20 dpi under hydroponic conditions. In contrast, nodulated vector control and LEC2AS plants appeared normal, and many fewer nodules formed on the root system . To express these findings in a quantitative way, the nodules were removed from the roots of the hydroponically grown plants and were separated into pink and senescent categories. Table 1 shows that the mean number of pink nodules was significantly lower for the LEC1AS plants than for the vector control and the LEC2AS plants. In contrast, the mean number of senescent nodules was significantly higher for the LEC1AS plants compared with the vector control andLEC2AS plants . The mean for the total number of nodules for the three plant groups, which included pink and senescent nodules,4x8ft rolling benches did not differ significantly . However, when the mean total nodule number was normalized to grams of root dry weight, the value was significantly higher for LEC1AS plants compared with vector control and LEC2AS plants; the vector control and LEC2AS plants did not differ significantly from each other . These results demonstrate that the LEC1AS plants produced more nodules, even though their overall root mass was less than the vector control and LEC2AS plants. We reasoned that if the nodules produced on LEC1AS plants senesced faster than vector control and LEC2AS plants, then more rhizobial colony-forming units would appear on agar medium after squashing the LEC1AS nodules. Moreover, we anticipated that the large pink nodules would also have an increased number of CFU. Nodules were collected, weighed, surface-sterilized, and crushed, and the resulting extracts were diluted and plated on RDM culture plates. LEC2AS, LEC2ST, and vector control plants all had similar numbers of culturable S. meliloti cells per mg of nodule tissue . In contrast, there were large mean levels of culturable S. meliloti cells in LEC1ST and especially in LEC1AS nodules. These results suggest that the excessive proliferation of the nodule tissue on LEC1ST and especially on the LEC1AS plants was accompanied by an extensive increase in the numbers of culturable bacteria. Both pink and senescent nodules contained large numbers of viable bacteria. RNA from LEC1AS roots with nodules contained detectable levels of the antisense-MsLEC1 transgene ; no endogenous sense mRNA was observed. The nodulated roots from the different LEC1AS plants showed considerable variability in the amount of accumulation of this transgenic RNA. We had found earlier that there was a correlation between plants that demonstrated moderate to severe developmental and reproductive abnormalities and those with low accumulation of MsLEC1-antisense RNA in nodulated roots .
Similarly, low levels of MsLEC1-antisense RNA were detected in nodules using Northern analysis. The transgenic plants were stably transformed, and different lines contained varying numbers and positions of transgene insertions , which may have contributed to variability in transgenic and endogenous lectin mRNA expression . Transcripts hybridizing to MsLEC1-sense mRNA were not detected in RNA isolated from nodules of LEC1AS plants . In situ hybridization analysis. Because of the difficulty in detecting MsLEC1 transcripts in nodules using Northern blot analysis, we performed in situ hybridization experiments on nontransgenic alfalfa nodules to get a better idea of the spatial expression pattern of this gene. Transcripts hybridizing to MsLEC1 were detected in alfalfa nodule meristems and adjacent cells of the invasion zone , whereas no transcripts were observed in the comparable cells of the sense controls . We also examined MsLEC1 expression in alfalfa roots and found that this lectin gene was expressed in the root apical meristem and also in cells of the elongation zone ; no transcripts were observed in the sense controls . It was difficult to evaluate the difference in the extent of MsLEC1 expression in individual uninoculated versus inoculated roots. The two sets of roots looked almost identical. For MsLEC2, essentially the same pattern of transcript localization was observed. Figure 6J illustrates an entire alfalfa nodule primordium 7 dpi; MsLEC2 mRNA was detected throughout the developing nodule using the WISH method. As the nodule matured, the signal became more concentrated in the cells of zones I and II, the nodule meristem and the invasion zone, respectively . More mature nodules showed the same pattern of MsLEC2 mRNA localization . MsLEC2 transcripts were also detected in the root meristems and adjacent regions and lateral root tips . Similar to the MsLEC1 results, there was no obvious difference in the amount of transcript observed in inoculated versus uninoculated roots. There was no signal detected in the nodule hybridized with the sense probe or in the comparable control for the root . To help confirm the spatial expression pattern of the soluble lectin genes in indeterminate nodules, we examined white sweetclover roots and nodules. This legume appears to have only a single copy of a putative seed lectin gene, termed MaLEC . This contrasts with alfalfa and Medicago truncatula, each of which has three different LEC genes . In white sweetclover nodules, MaLEC mRNA was detected in a 21-day-old nodule, in the cells of zones I and II, the nodule meristem and the invasion zone, respectively , in a pattern that was identical to that observed for alfalfa nodules . The nodule hybridized with the sense probe is depicted in Figure 6L; no signal was detected. Transcripts hybridizing to MaLEC were also detected in the main root tip and in lateral root meristems of both inoculated and uninoculated roots ; no transcripts were observed in the roots hybridized with the sense control .