Endopolyploidy Variation In Roots As Affected By Salinity And Phosphate Deficiency In Barley And Wheat

Endopolyploidy, which refers to different ploidy levels within an organism, is common among plants. Increased endopolyploidy is thought to play important roles in plant growth, development and adaptation to environmental stresses. However, little attention has been paid to reduced endopolyploidy. So far, much endopolyploidy adjustment has been learned from above-ground tissues of plants, limited information is available for root systems which are indispensable for land plants to acquire water and nutrients from soils, especially for the roots of economically important temperate cereals, such as wheat and barley. In this study, endopolyploidy levels were examined by using flow cytometry in different types and segments of roots of barley and hexaploid wheat, and effects of genome ploidy, moderate salinity and phosphate deficiency were also investigated. In addition, RNA sequencing and transcript levels of cell-cycle related genes in different root segments derived from P-sufficient and-deficient plants were monitored for the molecular mechanisms underlying endopolyploidy adjustment.Results showed that endopolyploidy levels varied among different root tissues and different root segments. Though the patterns of endopolyploidy were similar between barley and hexaploid wheat, barley presented higher endopolyploidy levels than hexaploid wheat in all the conresponding regions we measured. Furthermore, endopolyploidy levels were lower in lateral roots than in either primary or nodal roots, and also lower in the meristem/elongation zone of roots than in the mature zone in both barley and wheat.To find out what typies of root cells contribute to the endopolyploidy difference between lateral and nodal roots, cell size and nuclear size in the mature zone of three root types of barley was observed, and peripheral (cortical/epidermal) cells and stellar cells were physically separated and used for transcript analysis and ploidy measurements. It showed that the nuclear size was variable in both cortex and stele.The transcript levels of two marker genes which expressed predominantly in either peripheral cell layers (HvHKT2;1) or stellar cells (HvIPS2) showed a reasonable clean separation of peripheral cell layers from the stele. Also, it showed that the endopolyploidy level was higher in peripheral (cortical/epidermal) cells than that in stellar cells of nodal roots, but was similar in lateral roots in barley. Although cells in both cortical and stellar cells contribute to the higher endopolyploidy level in the mature zone, the lower endopolyploidy in lateral roots is responsible for the cortical cells as there was little endopolyploidy difference in the stelar cells between lateral and nodal roots. In addition, epidermal cells contribute relatively little to the higher endopolyploidy level in the cortical/epidermal cells observed.The difference in root endopolyploidy observed between barley and hexaploid wheat prompted us to examine effects of genome ploidies on root endopolyploidy. Results showed that the increase in the wheat genome ploidies from its two progenitors with lower ploidies decreased the endoployploidy level of primary roots, suggesting that the low endopolyploidy level in hexaploid wheat is largely due to its high genome ploidies. This negative correlation could also be applied to explain the endopolyploidy difference in roots between hexaploid wheat and barley, as both of them belong to the Triticeae family.To find out whether the endopolyploidy adjustment in roots is associated with plant adaptation to environmental stresses, two abiotic stresses, salinity and P deficiency, were examined for the effects on somatic cell ploidy levels of roots. Moderate salinity had no effects on the endopolyploidy levels in primary roots and their lateral roots, while slightly reduced the endopolyploidy level in the mature zone of nodal roots in barley. In addition, genotypic differences in the endopolyploidy level were observed between two barley genotypes. Sahara had a significantly higher endopolyploidy level across treatments in the basal zone of primary roots, mature zones of both primary and nodal roots than Clipper. In hexaploid wheat, it slightly reduced endopolyploidy levels and no genotypic difference existed between two wheat genotypes. P deficiency significantly reduced endopolyploidy levels in both lateral roots and the mature zone of primary roots, but not in the meristem/elongation zone of primary roots in barley. In addition, the average size of cortical and stelar cells in the mature zone of lateral roots and primary roots over two P treatments showed that the endopolyploidy reduction by P deficiency is less likely due to the change in the cell size. Taken together, salinity and P deficiency reduced endopolyploidy levels in a root type-or segment-specific manner.To understand the molecular mechanisms underlying the endopolyploidy difference between lateral and primary roots, and the endopolyploidy reduction by P deficiency, transcript levels of twelve cell cycle-related genes were examined. Transcript levels of the twelve cell cycle-related genes in different root segments of barley revealed that the expression of five out of eight HvCYC genes were higher in lateral roots than primary roots of barley, suggesting that the lower endopolyploidy level in lateral roots results from the modulation of multiple phases of the cell cycle and endocycle regulation differs between lateral and primary roots. Furthermore, comparisons of differently expressed genes (HvCCS52A1 and HvWEE1) in lateral and primary roots further support that the cell-cycle regulation in lateral roots differs from primary roots. In addition, RNA sequencing showed that cell cycle related genes involed in the regulation of endopolyploidy difference between primary and lateral root in barley, but they were not directly related to endopolyploidy reduction occurred by phosphate deficiency. Therefore, there should be existed other genes responsible for the molecular mechanisms underlying endopolyploidy reduction under phosphate stress.Our results provide new insights into the endopolyploidy variation in different root tissues and segments of two economically important cereals, and provide evidence that the endopolyploidy reduction occurs in the root system, suggesting that it is an integrated part of the endopolyploidy adjustment in plant growth and development and adaptation to some of environmental stresses. Therefore, it is necessary to take somatic ploidy variation into account for the functional analysis of root systems.