Manipulating male fertility in barley (Hordeum vulgare L.)

Formation of secondary wall thickening in the endothecium layer of anther is important for anther dehiscence and pollen release. In Arabidopsis this process has been shown to be regulated by R2R3-type MYB transcription factor MYB26 (Yang et al., 2007) and two NAC domain transcription factors, NAC Secondary Wall Promoting Factor 1 (NST1) and NST2 (Mitsuda et al., 2005). Characterisation of the regulation of endothecium development and anther dehiscence in agronomically important cereals such as barley will enable better understanding of pollen dispersal process in barley and other important crops. Moreover, in the longer term it may also facilitate identification of a stable sterile line for hybrid seed production in barley and potentially other cereals such as wheat. This project was aimed at identification and characterisation of the putative NST1 and NST2 orthologues in barley using reverse genetics approaches. The putative orthologous sequences in barley were identified by initially identifying the rice and Brachypodium putative sequences, prior to obtaining barley putative orthologues. The authenticity of the identified sequences was confirmed by comparison of their expression patterns and the level of synteny between Arabidopsis, rice, Brachypodium and barley sequences. These sequences were analysed by repression and overexpression in barley Golden Promise cultivar. SRDX repression of the putative sequences in barley resulted in reduction of fertility and associated reduction in plant height and spike size. The overexpression lines of HvNST1 and HvNST2 also showed reduction in fertility coupled with reduced spike size and plant height. Expression analysis in the barley transgenic lines suggested that the NST2 putative orthologue was regulated by NST1 and that MYB26 seemed to be upstream of these two genes. Further analysis is needed to confirm the nature of this interaction in barley. Moreover, this project explored the possibility of degradation of Arabidopsis Male Sterile 5 protein via the N-end rule proteolysis pathway. The preliminary results of this experiment suggested that the N-terminus of AtMS5 could be important in its degradation under in vitro conditions; however there needs to be further analyses to confirm this behaviour in vivo

Accurate staging of reproduction development in Cadenza wheat by non-destructive spike analysis

© 2020 The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. Wheat is one of the most important crops in the world; however, loss of genetic variability and abiotic stress caused by variable climatic conditions threaten future productivity. Reproduction is critical for wheat yield; however, pollen development is amongst the developmental stages most sensitive to stresses such as heat, cold, or drought. A better understanding of how anther and pollen development is regulated is needed to help produce more resilient crops and ensure future yield increases. However, in cereals such as wheat, barley, and rice, flowers form within the developing pseudostem and therefore accurate staging of floral materials is extremely challenging. This makes detailed phenotypic and molecular analysis of floral development very difficult, particularly when limited plant material is available, for example with mutant or transgenic lines. Here we present an accurate approach to overcome this problem, by non-destructive staging of reproduction development in Cadenza, the widely used spring wheat research variety. This uses a double-scale system whereby anther and pollen development can be predicted in relation to spike size and spike position within the pseudostem. This system provides an easy, reproducible method that facilitates accurate sampling and analysis of floral materials, to enable anther and pollen developmental research

Increased expression of the HvMALE STERILITY1 Transcription Factor Results in Temperature-Sensitive Male Sterility in Barley

Understanding the control of fertility is critical for crop yield and breeding, this is particularly important for hybrid breeding to capitalise upon the resultant hybrid vigour. Different hybrid breeding systems have been adopted, however these are challenging and crop specific. Mutants with environmentally-reversible fertility offer valuable opportunities for hybrid breeding. The barley HvMS1gene encodes for a PHD-finger transcription factor that is expressed in the anther tapetum, which is essential for pollen development and causes complete male sterility when over-expressed in barley. This male sterility is due at least in part to indehiscent anthers resulting from incomplete tapetum degeneration, failure of anther opening and sticky pollen, under normal growth conditions (15˚C). However, dehiscence and fertility is restored when plants are grown at temperatures >20˚C, or when transferred to >20˚C during flowering prior to Pollen Mitosis I, with transfer at later stages unable to rescue fertility in vivo. As far as we are aware this is the first report of thermo-sensitive male sterility in barley. This offers opportunities to understand the impact of temperature on pollen development and potential applications for environmentally-switchable hybrid breeding systems; it also provides a “female” male sterile breeding tool that does not need emasculation to facilitate backcrossing

Manipulating male fertility in barley (Hordeum vulgare L.)

Formation of secondary wall thickening in the endothecium layer of anther is important for anther dehiscence and pollen release. In Arabidopsis this process has been shown to be regulated by R2R3-type MYB transcription factor MYB26 (Yang et al., 2007) and two NAC domain transcription factors, NAC Secondary Wall Promoting Factor 1 (NST1) and NST2 (Mitsuda et al., 2005). Characterisation of the regulation of endothecium development and anther dehiscence in agronomically important cereals such as barley will enable better understanding of pollen dispersal process in barley and other important crops. Moreover, in the longer term it may also facilitate identification of a stable sterile line for hybrid seed production in barley and potentially other cereals such as wheat. This project was aimed at identification and characterisation of the putative NST1 and NST2 orthologues in barley using reverse genetics approaches. The putative orthologous sequences in barley were identified by initially identifying the rice and Brachypodium putative sequences, prior to obtaining barley putative orthologues. The authenticity of the identified sequences was confirmed by comparison of their expression patterns and the level of synteny between Arabidopsis, rice, Brachypodium and barley sequences. These sequences were analysed by repression and overexpression in barley Golden Promise cultivar. SRDX repression of the putative sequences in barley resulted in reduction of fertility and associated reduction in plant height and spike size. The overexpression lines of HvNST1 and HvNST2 also showed reduction in fertility coupled with reduced spike size and plant height. Expression analysis in the barley transgenic lines suggested that the NST2 putative orthologue was regulated by NST1 and that MYB26 seemed to be upstream of these two genes. Further analysis is needed to confirm the nature of this interaction in barley. Moreover, this project explored the possibility of degradation of Arabidopsis Male Sterile 5 protein via the N-end rule proteolysis pathway. The preliminary results of this experiment suggested that the N-terminus of AtMS5 could be important in its degradation under in vitro conditions; however there needs to be further analyses to confirm this behaviour in vivo