Differences in total root length, surface and volume density, average root diameter, and root tips and fork densities were analyzed using a split-plot design with genotypes as main plots and depth as subplot. This is a conservative statistical analysis because it reduces the df for genotype from 3 to 1. Therefore, we also compared the two same pairs of genotypes using statistical contrasts in an ANOVA including all four genotypes. To account for the inability to randomize depths, we used a conservative estimate of the df for subplots and for the interaction between subplot and main plot. Conservative df were calculated by dividing their df by the number of subplots. This strategy is similar to that used for repeated measures in time and does not affect comparisons among main plots , which are the main objective of this study. Homogeneity of variance and normality of the residuals was confirmed for all the individual ANOVAs performed at each depth for all parameters. When necessary, data was transformed using power transformations to satisfy ANOVA assumptions.The experiments were performed in 13 L hydroponic tanks containing the nutrient solution. Twenty-four seedlings were placed in each tank in a six by four pattern. All genotypes were included in one tank and, if necessary, multiple tanks were used as replications. In the experiments to study the effect of different nitrogen sources and concentrations ,vertical growing systems seedlings were grown in normal growth solution for seven days and then transferred to four separate tanks with each of the four nitrogen sources for 10 days .
Roots were measured at 22 DAG. Each tank included 6 replications of each of the four genotypes organized in a completely randomized design. The results were analyzed in a 2 x 2 factorial ANOVA with distal and proximal 1RS regions as factors and wheat or rye chromosome segments as levels. For the analysis of distances between the first lateral roots and the RAM, the four genotypes were grown in a tank with normal nitrogen conditions in a completely randomized design . Lines carrying distal rye or wheat segments were compared using a t-test. Chascomús, Buenos Aires Argentina: The CaCl2 from the germination tank was replaced by nutrient solution on the 4th day. On the 5 th day, plants were transferred to 350 mL pots containing nutrient solution, with each pot being a replicate. Pots were rotated every two days to ensure that they occupied different positions within the growth chamber. For the root elongation time course, the length of the second longest seminal root was measured daily four hours after the start of the light period, starting 6 DAG. Within each experiment, data was analyzed as repeated measures . A combined ANOVA was performed using experiments as blocks.The NBT staining experiments were performed using 5 cm root sections excised from the second longest root of 1RS and 1RSRW plants 17 DAG. This root segment was placed for 90 min in a 0.1 mg mL-1 NBT solution dissolved in 200 mM potassium phosphate buffer, pH 7.6, in darkness. For the DCF-DA staining similar root segments were placed in the same buffer supplemented with 10 µM DCF-DA for 60 min, in darkness. Roots segments stained with NBT or DCF-DA were washed in the same buffer for 30 min and placed on a slide. Roots were observed using a Zeiss Discovery.V20 stereo microscope equipped with a coaxial fluorescence mechanism. Pictures were obtained with an Axiocam 512 color . The images were processed with the ImageJ software obtaining a longitudinal profile of color and fluorescence intensities.
These experiments were performed in Chascomús. Previous studies have shown an association between the introgression of the rye 1RS arm in wheat and improved resistance to water stress . In three of these studies, the 1RS.1BL lines showed increased root biomass compared to the non-1RS control lines in large pot or sand-tube experiments. However, these differences were not validated in the field. In this study we showed that differences in grain yield and biomass between plants carrying a complete 1RS translocation and NILs with an introgressed distal wheat chromosome segment are associated with differences in total root length density and average root diameter in the field. Field excavations of the four different 1RS NILs provided an opportunity to visualize the differences in their root systems and to quantify these differences using horizontal soil cores at consistent depths. This experiment confirmed the hypothesis that the 1RSxR lines have a higher root density throughout the soil profile, with roots that reach deeper in the soil than the 1RSxW lines . The more extensive root system of the 1RSxR lines relative to the 1RSxW lines may have contributed to their better tolerance to drought and water logging conditions in the experiments presented in this study , and to the higher carbon isotope discrimination and increased stomatal conductance values detected in a previous study . Through their deeper root system, the 1RSxR plants can access more stored soil moisture and nutrients, keep their stomata open longer, and generate additional photosynthetic products and biomass than the 1RSxW plants. However, we cannot rule out the possibility that the genes in the distal 1BS introgression may have a more direct effect on aerial biomass or on other anatomical and/or physiological root differences known to impact tolerance to water logging and drought . The differences in root depth observed between the Hahn 1RSxR and 1RSxW NILs in the field were paralleled by drastic changes in seminal root length in hydroponic cultures . These differences were robust across experiments and were detected with different nitrogen sources and concentrations .
We hypothesize that these early differences in seminal root length may have contributed to the observed differences in total root length density observed in the deepest soil core samples in the field . The early and consistent differences in root growth under controlled conditions provided the opportunity to study the process in detail. During the first week of development, root growth occurred at the same rate for both genotypes, suggesting that the differences were not primarily associated with embryonically determined differences in root elongation. Instead, differences in root growth consistently manifested during the second week across multiple experiments. The growth rate of the seminal roots of the 1RSxW plants gradually decreased during the second and third week, to come close to zero by the end of the third week, whereas growth continued in the 1RSxR plants . The consistent timing of these events suggests that these changes are developmentally regulated. The growth arrest of the seminal roots in the 1RSxW plants was accompanied by the proliferation of lateral roots in close proximity to the RAM, suggesting important changes in the RAM. The RAM consists of a quiescent center surrounded by stem cells that generate new daughter cells, which undergo additional divisions in the proximal region of the meristem and differentiatein the transition zone . At a cellular level, a balance between cell proliferation and cell elongation/differentiation determines root growth rate . The arrest of the growth of seminal roots in 1RSxW plants suggests a modification in cell proliferation and/or cell elongation/differentiation. Additional studies will be required to determine if this arrest involves changes in the QC and/or modifications in the root regions adjacent to the meristem. In any case,outdoor vertical plant stands the dramatic reduction in seminal root growth and increased lateral root proliferation close to the RAM argues for an early developmental program switch in the regulation of the RAM in the 1RSxW plants. The transition from cell proliferation to cell elongation and differentiation and the subsequent development of lateral roots depends on the distribution of ROS along the root axis, specifically on the opposing gradients of superoxide and hydrogen peroxide.
Superoxide is predominant in dividing cells in the meristematic zone, while hydrogen peroxide is predominant in elongated cells in the differentiation zone . The balance between these ROS modulates the transition between root proliferation and differentiation zones. Seventeen days after germination, the apical region of 1RS seminal roots showed opposing gradients of superoxide and hydrogen peroxide characteristic of elongating roots . A different ROS distribution was detected in the arrested 1RSxW roots, where superoxide was restricted to the distal ~700 m and increased levels of DCF-DA fluorescence were detected between 250-950 m in the cell proliferation zone . The contrasting patterns of ROS distribution reflect the major developmental changes that differentiate the seminal roots of the 1RS and 1RSxW genotypes. Studies in Arabidopsis have shown that changes in ROS distribution can be triggered by the altered expression of major genes that control the size of the meristematic zone. These genes include UPBEAT1 , a basic helix-loop-helix transcription factor that regulates the meristematic zone size by restricting H2O2 distribution in the elongation zone . In addition, ROOT MERISTEM GROWTH FACTOR 1and the transcription factor RGF1 INDUCIBLE TRANSCRIPTION FACTOR 1that mediates RGF1 signaling can modulate the distribution of ROS along the root developmental zones leading to enhanced stability of PLETHORA2 . Reduced expression of PLETHORA in the root apical region or changes in its distribution have been associated with impaired root growth. To test if these Arabidopsis results are applicable to wheat, we are initiating expression studies of these genes in the 1RSxR and 1RSxW lines. It remains unknown if the differential pattern of ROS distribution in the roots of the 1RSxW plants is the result of changes in the wheat homologs of these central developmental genes or a more direct effect on genes affecting the redox balance in different developmental root zones. The differences in superoxide and hydrogen peroxide distribution between the seminal roots of the 1RSRW and 1RS plants were measured after the arrest in root growth . Therefore, we currently do not know if the changes in ROS distribution are a cause or consequence of the changes observed in root growth and lateral root proliferation close to the RAM. Strawberry frigoplants cv. Elsanta were cultivated in hydroponics. The variation in N-form started when the plants switched over from the vegetative to the generative growth phase. During the vegetative phase, all plants were fed with NO3-N. After changing to the generative stage, plants were divided into three different nutrition treatments, containing two ammonium treatments and one nitrate treatment. The solution pH was stabilized to pH 3.5 and 5.5 by pH-stat. The nutrient solution was changed every 3 d.The concentrations of the main organic acids in the fruits were measured using high performance liquid chromatography . Seeds of E. camaldulensis were germinated on acid-washed and sterilized sand in a growth chamber . Seedlings were watered daily with a nutrient solution and grown for 3 to 4 months. After seedlings were cultured hydroponically for 5 days, roots of seedlings were treated with 0.35 mM CaCl2 solution containing 0 or 1 mM AlCl3 for 24 h. The 5 mm apical portions of roots were excised, freeze-thawed, and centrifuged on an ultrafilter to obtain soluble compounds from root-tip cells. Al-binding compounds which were soluble in the form of complex with Al were separated by gel-filtration chromatography. The cellular extract added with Al was subjected to a gel-filtration column to separate the complexes of Al-binding compounds with Al from free Al. The Al concentration of each fraction was determined with an inductively coupled plasma atomic emission spectrometer . In addition, Al-binding compounds which became insoluble in the form of complex with Al were separated by HPLC equipped with a reversed-phase column. The cellular extract added with or without Al was subjected to HPLC analysis. Chromatographic peaks whose area was decreased by the addition of Al were judged to be those of Al-binding compound. When Al was added to the cellular extract from roots cultivated without Al and the extract was subjected to the gel-filtration column, one peak was observed by monitoring the Al concentration in each fraction. Two peaks were found when the roots were treated with 1 mM Al. These results indicate that roots of E. camaldulensis contain Al-binding compounds which are soluble in the form of complex with Al, and the compounds increase with Al treatment. We also measured organic acids in each fraction, and found that the peak of citrate corresponded to that of the Al-complex. The increase of Al-binding compound can be explained partly by the increase of citrate.