When necessary, data was transformed using power transformations to satisfy ANOVA assumptions. Hydroponic experiments were performed in growth chambers at 22-23 °C with a photoperiod of 16 h light / 8 h dark provided by fluorescent lights supplemented with incandescent lighting. In all experiments, grains were imbibed at 4°C for four days, after which they were placed at room temperature. Once most of the grains had germinated and the coleoptiles had emerged, healthy seedlings were transferred to a mesh suspended on water or CaCl2 . Four days later, healthy seedlings were transferred to tanks with growth solutions 6Mo7O24.4H2O 0.05 M, CuSO4.5H2O 0.5 M, ZnSO4.7H2O 1 M, MnSO4.H2O 1 M, FeEDTA 0.1 mM, Ca2 1.0 mM, 2SO4 1.0 mM. After removing the grain, seedlings were wrapped at the crown with foam and inserted in holes pre-cut in a foam core board placed on top of the solution. Nutrient solution was changed two to three times a week for the duration of the experiment. Particular details of the methods used in the two laboratories are described below: Davis, CA, USA: 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 , 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, round plastic planter 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. 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, 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,round plastic plant pot 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. Wheat is a crop of major importance and together with other staple cereals supply the bulk of calories and nutrients in the diets of a large proportion of the world population . A major focus of wheat breeders has been grain protein concentration as it affects bread- and pasta-making quality, but micro-nutrient improvement has received less attention. Approximately half of the world’s population suffers from Fe and/or Zn deficiencies and millions of children suffer from protein-energy malnutrition . As such, the improvement of nutritional quality of wheat could benefit the nutritional status of millions of people. A common agronomic practice to increase grain protein concentration is the use of N fertilization. However, this practice is expensive and excess fertilizer run-off is a potential environmental contaminant .
A substantial percentage of the N in wheat grain is supplied by amino acids remobilized from vegetative tissue . Much of this N content is derived from proteins that are disassembled and recycled during the leaf senescence stage of development . Likewise, Fe and Zn have been shown to be remobilized from vegetative tissues in several plants , although the specific sources are unknown. Zinc fertilization has been a successful strategy to improve wheat grain Zn concentration , and improvement in the partitioning or remobilization of Zn to grain could make fertilization efforts more efficient. Wheat grain with higher Zn concentration has been demonstrated to produce more vigorous crops . Thus, breeding or transgenic approaches that result in plants with increased partitioning of minerals to grain could be useful for both nutritional bio-fortification and reduced fertilizer application. Chromosome 6B from wild emmer wheat was identified as a potential source of genetic variation for grain protein , Zn, and Fe concentration . A quantitative trait locus for grain protein concentration was mapped on chromosome arm 6BS and later mapped as a single Mendelian locus, Gpc-B1 . In near-isogenic lines of this locus, increased grain protein was associated with the increased remobilization of amino acids from the flag leaf , higher grain Fe and Zn concentrations , and accelerated leaf yellowing, indicating accelerated senescence . A NAC transcription factor, NAM-B1, was identified as the causal gene for Gpc-B1 by positional cloning . Other members of the NAC family are known to regulate developmental processes , including leaf senescence . In transgenic wheat NAM RNA interference lines in which NAM-B1 and its homeologous genes had decreased expression, leaf yellowing was delayed, and grain protein, Fe, and Zn concentrations were greatly decreased . These results, together with higher N, Fe, and Zn concentrations in RNAi line flag leaves at maturity, suggested a role for NAM-B1 homeologues in the remobilization of N compounds, Fe, and Zn. However, without taking organ mass, nutrient concentrations at prior time points, and total nutrient accumulation of other organs into account, this model could not be confirmed. In addition, the body of literature does not contain sufficient data regarding sources of grain minerals to support the idea that remobilization alone could account for the differences observed. Because a whole-plant partitioning profile has not been undertaken in plants differing in NAM-B1 expression, it is currently unclear whether this gene directly affects remobilization , alters partitioning of nutrients within the plant, alters total plant uptake of these nutrients, or influences a combination of these processes.