The present study was conducted to address the dosage effect of 1RS translocation in bread wheat. We used wheat genotypes that differ in their number of the 1RS translocations in a spring bread wheat ‘Pavon 76’ genetic background. For generating F1 seeds, Pavon 1RS.1AL was the preferred choice due to its better performance for root biomass than other 1RS lines . Here, we report the dosage effect of a 1RS chromosome arm on the morphology and anatomy of wheat roots. The results from this study validate previous results of the presence of genes for rooting ability on the 1RS chromosome arm. This study also provides evidence for presence of genes affecting root anatomy on 1RS. From previous chapters of this dissertation and earlier studies , it was clear that there was a gene present on 1RS chromosome arm which affects root traits in bread wheat. But there was no report on the chromosomal localization of any root anatomical trait in bread wheat. The purpose of this study was to look for variation in root morphology and anatomy among different wheat genotypes and then determine how these differences are related to different dosages of 1RS in bread wheat. During this study, we came to some very interesting conclusions: 1) F1 hybrids showed a heterotic effect for root biomass and there was an additive effect of the 1RS arm number on root morphology of bread wheat; 2) There was a specific development pattern in the root vasculature from top to tip in wheat roots and 1RS dosage tended to affect root anatomy differently in different regions of the seminal root. Further, the differences in root morphology,hydroponic gutter and especially anatomy of the different genotypes have specific bearing on their ability to tolerate water and heat stress. The effect of number of 1RS translocation arms in bread wheat was clearly evident from their averaged mean values for root biomass. RA1 and RAD4 were ranked highest while R0 ranked at the bottom .

These results supported the previous studies on the performance of wheat genotypes with 1RS translocation where 1RS wheats performed better in grain yield but similar for shoot biomass . Genotype RD2 performed slightly better than R0 for root biomass because of its poor performance in one season otherwise it showed better rooting ability in the other three seasons. Here, all the genotypes with 1RS translocations showed higher root biomass than R0 which carried a normal 1BS chromosome arm. Data in this study suggested two types of effects of 1RS on wheat roots. First, an additive effect of 1RS, there was increase in root biomass with the increase in 1RS dosage from zero to two and then to four . Second was a heterotic effect of 1RS on root biomass and shoot biomass. MPH and HPH of the F1 hybrid were higher for root biomass than for shoot biomass . This further explained the more pronounced effect of 1RS on root biomass than shoot biomass. Significant positive heterosis was observed for root traits among wheat F1 hybrids and twenty seven percent of the genes were differentially expressed between hybrids and their parents . The possible role of differential gene expression was suggested to play a role in root heterosis of wheat and other cereal crops . In a recent molecular study of heterosis, it was speculated that upregulation of TaARF, an open reading frame encoding a putative wheat ARF protein, might be contributing to heterosis observed in wheat root and leaf growth There is large void in root research involving study of root anatomy in wheat as well as other cereal crops. Most of the anatomical literature is either limited to root anatomy near the base of the root or near the root tip in young seedlings . There is still a general lack of knowledge about the overall structure and pattern of whole root vasculature during later stages of the growth in cereals especially in wheat. In the present study, root anatomical traits were studied in the primary seminal root of different wheat genotypes containing different dosages of 1RS translocation arms at mid-tillering stage .

Root sections were made from three regions along the length of the root, viz. top of the root, middle of the root and root tip, to get an overview of the complete structure and pattern of root histology relative to differences in 1RS dosage. Comparison of different regions of root of a genotype showed a transition for metaxylem vessel number and CMX area from higher in top region of the root to a single central metaxylem vessel in the root tip. Diameter of the stele also became narrower towards the root tip as the plant roots grow into deeper layers of soil. In the root tip only central metaxylem vessel diameter and area were traceable as other cell types were still differentiating. This developmental pattern was consistent across the different wheat genotypes used in this study. Interestingly, there was variation in timing for the transitions in root histology among genotypes and this variation was explained by dosage of 1RS arm in bread wheat. RD2 and RAD4 transitioned earlier from having multiple metaxylem vessels and a larger stele to a single, central metaxylem vessel and smaller stele than did R0 and RA1. In the top region, all the root traits were significantly different among genotypes except average CMX vessel diameter and CMX vessel number . Here, the average CMX diameter was calculated from the average of diameters of all the CMX number of that subsequent genotype and hence, the number of CMX vessels, was different in each genotype so was the total CMX vessel area. Interestingly, all the root traits in the top region showed negative slope in regression analysis and most of them were significant especially stele diameter, total CMX vessel area, and peripheral xylem pole number. Variation in all the traits was explained by number of 1RS dosages in wheat genotypes and root traits were smaller with higher number of 1RS dosage . Significant positive correlation among almost all the root traits from topregion and mid-region of the roots suggested their interdependences in growth and development. Root diameter could not be measured for all the replicates of each genotype because of the degeneration and mechanical damage to the cortex and epidermis.

Earlier, a study on the rate of cortical death in seminal roots was investigated in different cereals.In the root tip, only two traits, CMX vessel area and CMX vessel diameter, were traceable because of the status of root tip development . Negative slope and significant R2 value in regression analysis explained the effect of 1RS dosage on the CMX vessel area and CMX vessel diameter. This suggested narrow metaxylem vessels with increase in 1RS dosage . In roots, central metaxylem vessel is the first vascular element to be determined and differentiate . Here, serial cross sections of the root tips also confirmed it as the first differentiated vascular element in wheat. The other vascular components differentiate thereafter in relation to first formed metaxylem vessel . Feldman first reported that all the metaxylems were not initiated at the same level. Root morphology and root architecture are responsible for the water and nutrient uptake while in root anatomy, xylem vessels are essential for their transportation to the shoots to allow continued photosynthesis. Variations in xylem anatomy and hydraulic properties occur at interspecific, intraspecific and intraplant levels . Variations in xylem vessel diameter can drastically affect the axial flow because of the fourth-power relationship between radius and flow rate through a capillary tube, as described by the Hagen–Poiseuille law . Thus, even a small increase in mean vessel diameter would have exponential effects on specific hydraulic conductivity for the same pressure difference across a segment . Xylem diameters tend to be narrower in drought tolerant genotypes ,u planting gutter and at higher temperature . Smaller xylem diameters pose higher flow resistance and slower water flow which helps the wheat plant to survive water stressed conditions. Richards and Passioura increased the grain yield of two Australian wheat cultivars by selecting for narrow xylem vessels in seminal roots. The results of this study showed that the presence of 1RS in bread wheat increased the root biomass and reduced the dimensions of some root parameters especially the central metaxylem vessel area and diameter in the root tip as well as in the top of the root . Manske and Vlek also reported that wheat genotypes with 1RS translocated chromosome arm had thinner roots and higher root-length density compared with normal wheat with 1BS chromosome arm under field conditions. These results might suggest higher root number or extensive root branching in 1RS translocation wheats. Among 1RS translocation wheats, significant association was observed between root biomass and grain yield under well-watered and droughted environments . Narrow metaxylem vessels and higher root biomass provide 1RS translocation wheats with better adaptability to water stress and make them better performers for grain yield. Plant development is particularly sensitive to light, which is both the energy source for photosynthesis and the regulatory signal . Upon germination in the dark, a seedling undergoes a developmental program named skotomorphogenesis, which is characterized by elongated hypocotyl, closed cotyledon, apical hook, and short root. Exposure to light promotes photomorphogenesis, which is characterized by short hypocotyl, open cotyledon, chloroplast development and pigment accumulation . In addition to light, photomorphogenesis is also regulated by several hormones, including brassinosteroid , auxin, gibberellin and strigolactone .

The molecular mechanisms that integrate the light and hormonal signals are not fully understood. Light signal is perceived by photoreceptors, which regulate gene expression through several classes of transcription factors . Downstream of photoreceptors, the E3 ubiquitin ligase COP1 acts as a central repressor of photomorphogenesis . COP1 targets several transcription factors for proteasome-mediated degradation in the dark . Light-activated photoreceptors directly inhibit COP1’s activity, leading to the accumulation of the COP1- interacting transcription factors, such as HY5 , BZS1, and GATA2, which positively regulate photomorphogenesis . Recent studies have uncovered mechanisms of signal crosstalk that integrate light signaling pathways with BR, GA, and auxin pathways . The transcription factors of these signaling pathways directly interact with each other in cooperative or antagonistic manners to regulate overlapping sets of target genes . BR has been shown to repress, through the transcription factor BZR1, the expression of positive regulators of photomorphogenesis, including the light-stabilized transcription factors GATA2 and BZS1 . BZS1 is a member of the B-box zinc finger protein family, which has two B-box domains at its N terminus without any known DNA binding domain . It is unclear how BZS1 regulates gene expression. Recent studies have shown that SL inhibits hypocotyl elongation and promotes HY5 accumulation in Arabidopsis plants grown under light , but the molecular mechanisms through which SL signaling integrates with light and other hormone pathways remain largely unknown. Immunoprecipitation of protein complexes followed by mass spectrometry analysis is a powerful method for identifying interacting partners and post translational modifications of a protein of interest . In particular, research in animal systems has shown that combining stable isotope labeling with IP-MS can quantitatively distinguish specific interacting proteins from non-specific background proteins . Stable isotope labeling in Arabidopsis has been established as an effective method of quantitative mass spectrometry ; however, combination of SILIA with IP-MS has yet to be established. To further characterize the molecular function of BZS1, we performed SILIA-IP-MS analysis of the BZS1 protein complex, and identified several BZS1-accociated proteins. Among those are COP1, HY5, and BZS1’s homologs STH2/BBX21 and STO/BBX24. We further showed that BZS1 directly interacts with HY5, and positively regulates HY5 RNA and protein levels. Genetic analysis indicated that HY5 is required for BZS1 to inhibit hypocotyl elongation and promote anthocyanin accumulation. In addition, BZS1 is positively regulated by SL at both transcriptional and translational levels. Plants over expressing a dominant-negative form of BZS1 show an elongated-hypocotyl phenotype and reduced sensitivity to SL, similar to the hy5 mutant. Our results demonstrated that BZS1 acts through HY5 to promote photomorphogenesis and is a crosstalk junction of light, BR and SL signals. This study further advances our understanding of the complex network that integrates multiple hormonal and environmental signals.