GSE112296
Analysis of Brachypodium miRNA targets: Evidence for diverse control during stress and conservation in bioenergy crops
Background: Since the proposal of Brachypodium distachyon as a model for the grasses over 500 Bdi-miRNAs have been annotated in miRBase making Brachypodium second in number only to rice. Other monocots, such as switchgrass, are completely absent from the miRBase database. While a significant number of miRNAs have been identified which are highly conserved across plants, little research has been done with respect to the conservation of miRNA targets. Plant responses to abiotic stresses are regulated by diverse pathways many of which involve miRNAs; however, it can be difficult to identify miRNA guided gene regulation when the miRNA is not the primary regulator of the target mRNA. Results: To investigate miRNA target conservation and stress response involvement, a set of PARE (Parallel Analysis of RNA Ends) libraries totaling over 2 billion reads was constructed and sequenced from Brachypodium, switchgrass, and sorghum representing the first public release of degradome data from the latter two species. Analysis of this data provided not only PARE evidence for miRNA guided cleavage of over 7,000 predicted target mRNAs in Brachypodium, but also evidence for miRNA guided cleavage of over 1,000 homologous transcripts in sorghum and switchgrass. A pipeline was constructed to compare RNA-seq and PARE data made from Brachypodium plants exposed to various abiotic stress conditions. This resulted in the identification of 44 miRNA targets which exhibit stress regulated cleavage. Time course experiments were performed to reveal the relationship between miR393ab, miR169a, miR394ab, and their respective targets throughout the first 36 hours of the cold stress response in Brachypodium. Conclusions: Knowledge gained from this study provides considerable insight into the degradomes and the breadth of miRNA target conservation among these three species. Additionally associations of a number of miRNAs and target mRNAs with the stress responses have been revealed which will aid researchers in developing stress tolerant transgenic crops. Overall design: Libraries were constructed from tissue samples from Brachypodium, sorghum, and switchgrass plants as well as above ground tissue which had been exposed to various stress conditions. Two biological replicates were constructed for each library type. Control libraries were constructed in addition to stress samples. Brachypodium  Bd21  seeds  were  germinated  in  soil  and  the  plants  were  grown  in  a  growth  chamber  at  a  constant  20°C,  under  a  20  h  light  350  µE·m-2·s-1,  4  h  dark  cycle.  On  day  21,  10  hours  after  light  started,  plants  were  subjected  to  various  stress  treatments.  For  heat  stress,  plants  were  placed  at  40°C.  For  drought  stress,  plants  were  removed  from  soil  and  the  roots  were  dried  with  paper  towels.  For  submergence  stress,  plants  were  submerged  completely  with at  least  5  cm  of  tap  water  covering  the  top  of  the  plants.  For  cold  stress,  plants  were  placed  at  4°C.  For  stress  libraries,  above  ground  tissue  was  sampled  after  12  hours  of  stress;  for  the  cold  stress  time  courses,  samples  were  taken  at  0,  1,  2,  3,  6,  12,  24,  and  36  hours.  Switchgrass  (AP13)  was  grown  under  greenhouse  conditions  with  an  average  day  temperature  of  26°C  and  an  average  night  temperature  of  20°C.    Stress  treatments  were  carried  out  two  months  after  propagation  from  cuttings  and  the  second  and  third  leaf  blades  from  the  top  were  sampled.  After  germination,  Sorghum  bicolor  (BTx623)  seedlings  were  transferred  to  soil  in  a  growth  chamber.  After  14  days  of  growth  (12  hours  light  at  28°C,  12  hours  dark  at  25°C),  cold  treatments  were  carried  out  and  the  above-ground  tissue  was  harvested.  Switchgrass  and  sorghum  cold  treatments  at  4°C  were  performed  for  24  hours.  For  switchgrass  drought  experiments,  the  drought  sample  was  harvested  11  days  after  watering  was  stopped.  Plants  subjected  to  recovery  were  then  watered,  and  on  the  12th  day  they  were  sampled.