Effect of high leaf temperature and nitrogen concentration on barley (Hordeum vulgare L.) photosynthesis and flowering

The response of plants to abiotic stress factors is a major determinant of the growth and yields of crops. In this study the effects of two separate but related abiotic stress factors on elite spring barley cultivars (Horedeum vulgare ) were studied; the effects of high leaf temperatures (Tleaf) on photosynthesis rates, and the effects of high nitrogen supply on photosynthesis rates and flowering. A novel method was developed for precisely controlling Tleaf within ± 0.2oC of the set temperature. These experiments confirmed the results of others that increasing Tleaf above 36.0oC for 3 hours severely impaired light saturated CO2 assimilation rates (Asat) by irreversibly suppressing the activity of the C3 cycle by >80%. This suppression was not attributable to stomatal closure, the generation of ROS, or an increase in photorespiration; instead the data were consistent with the hypothesis that limitations imposed by low chloroplast ATP levels. Measurements on whole leaf ATP levels in the light and dark of control and heat stressed leaves, however, were equivocal. Whole leaf ATP levels of light adapted leaves increased with Tleaf whereas the levels in dark adapted leaves initially decreased but increased again above 38oC; most importantly, the difference – an estimate of chloroplast ATP levels - increased with Tleaf, an observation that is not consistent with the hypothesis. The effects of high Tleaf was assessed on plants grown in soil and hydroponic solutions over a range of N-supply. Similar responses were observed regardless of the nitrogen status of the plants. Surprisingly, the unit leaf area (ULA) photosynthesis rates of control (not heat stressed) attached leaves doubled when plants were grown in 16 mM N compared with 0.6 mM N (commensurate with arable soils); detailed analysis of CO2 Assimilation vs. Internal CO2 concentration (A/Ci) curves showed the carboxylation coefficient (ϕCO2) increased suggesting the ULA capacity of the C3 cycle had been boosted and this correlated well with ULA protein levels. It seems there is a good prospect, therefore, for boosting ULA photosynthesis rates, and hence grain yield, by increasing plant N-status above that currently used in arable production. Increasing N-supply to these levels, however, has detrimental effects on the morphology and development of barley. Increasing N-supply above 0.3 mM induced tillering (increased resource sink strength) as well as yield; above 1.6 mM, however, yields began to decline, flowering was delayed, although tillering (vegetative growth) continued to proliferate. At the highest levels used (16 mM) floweing was completely suppressed; the crown meristem underwent a vegetative to reproductive transition but stem elongation was incomplete – plants rarely progress beyond the 3 node stage. A series of transcript profiling experiments were conducted to establish the mechanisms by which high N-status suppressed flowering. Analysis of the transcriptional activity of key components of the flowering pathway in leaves, coupled with observations on floral spike development suggested flowering was triggered an initiated the development of the inflorescence at the crown meristem, but high N inhibits the development of the floral primordia. A RNA-Seq experiment was undertaken to determine the transcriptome profiles of 2-3 node stage floral primordia in plants grown in 16 mM and 0.64 mM N-supply. These studies were hampered by the poor level of annotation of published barley sequences, but none-the-less several interesting candidate sequences, including a homologue of the Arabisopsis flowering gene AtAPETELLA2, were strongly down regulated; these results from these experiments are discussed in detail. Studies were also undertaken to manipulate sink strength in barley plants by reducing the number of tillers either mechanically (removal) or using ‘uniculm’ mutants from the Bowman barley accession lines. These experiments have proved to be challenging and progress has been slow; a discussion is provided on how these experiments may be completed. The ultimate goal of this project is to develop barley lines with an optimized sink strength (tiller number) that will not trigger excessive vegetative growth when plant N-status is high. This should lead to the retention of N in the leaves of the main culm leading to higher ULA photosynthesis rates and hence higher yields. To achieve this, however, these plants will have to be further manipulated so that high plant N-status does not suppress flower development.