Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

Background Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. Results We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

The individual copy number of genes localised in the plastid genome in subsequent days of isolated microspore culture of 'Jersey' and 'Mercada' cultivars in 'Jersey' and 'Mercada' cultivars. c The relative expression profile of Polγ (Organellar DNA polymeraseI) gene.Graphs show mean values of n ≥ 3 with SD in a and b or SEM in c.Relative expression level normalised to 43dC of cv.'Jersey'.An asterisk presents a value significantly different between cultivars at a certain day of culture.A hash indicates a value significantly different from the preceding day of culture within cultivar (Tukey's test, P < 0.05).A $ indicates value significantly different from the calculated average plastome copy number at a particular day of culture within cultivar (Student's t-test, P < 0.05).Red lines show the average copy numbers calculated from individual copies of presented genes.LSClong single copy, IRinverted repeat, SSCshort single copy, MLmid-to-late microspore, PMpre-treated microspores, dCday of culture.

Figure S7. The normalized expression level of genes important for plastid biogenesis during embryo formation and regeneration of androgenic plants.
Graphs show mean values of n ≥ 3 with SEM for expression level normalised to reference genes.An asterisk presents a value significantly different between 'Jersey' and 'Mercada' cultivars (t-Student test, P < 0.05).MLmid-to-late microspore, PMpre-treated microspores, dCday of culture.

Figure S2 .
Figure S2.The relative expression profile of DPD1 gene encoding Mg 2+-dependent organelle exonuclease during microspore development of cvs.'Jersey' and 'Mercada'.Graph shows mean values of n ≥ 3 with SEM.Relative expression level normalised to E microspores of cv.'Jersey'.An asterisk presents a value significantly different between cultivars at a certain day of microspore development.A hash indicates a value significantly different from the preceding day of microspore development within cultivar (Tukey's test, P < 0.05).Stages of pollen development: E early, EMearly-mid, MLmid-to-late.

Figure S3 .
Figure S3.Plastid DNA content during regeneration of androgenic plants of cvs.'Jersey' and 'Mercada'.a Average plastome copy number in relation to nuclear genome in subsequent days of regenerating plants of 'Jersey' and 'Mercada' cultivars.b The individual copy number of genes localised in the plastid genome in subsequent days of isolated microspore culture of 'Jersey' and 'Mercada' cultivars in 'Jersey' and 'Mercada' cultivars.c The relative expression profile of Polγ (Organellar DNA polymeraseI) gene.Graphs show mean values of n ≥ 3 with SD in a and b or SEM in c.Relative expression level normalised to 43dC of cv.'Jersey'.An asterisk presents a value significantly different between cultivars at a certain day of culture.A hash indicates a value significantly different from the preceding day of culture within cultivar (Tukey's test, P < 0.05).A $ indicates value significantly different from the calculated average plastome copy number at a particular day of culture within cultivar (Student's t-test, P < 0.05).Red lines show the average copy numbers calculated from individual copies of presented genes.LSClong single copy, IRinverted repeat, SSCshort single copy, MLmid-to-late microspore, PMpre-treated microspores, dCday of culture.

Figure S4 .
Figure S4.The expression profiles of genes related to chloroplast differentiation during regeneration of plants of cvs.'Jersey' and 'Mercada'.Gene encoding transcription factor HY5 and involved in synthesis (PGP1) and docking (RABA5e) of thylakoids in 'Jersey' and 'Mercada' cultivars.Graphs show mean values of n ≥ 3 with SEM.Relative expression level normalised to 43dC of cv.'Jersey'.An asterisk presents a value significantly different between cultivars at a certain day of culture.A hash indicates a value significantly different from the preceding day of culture within cultivar (Tukey's test, P < 0.05).dCday of culture.

Figure S5 .
Figure S5.The plastids observed in converting embryos on 46dC of cvs.'Jersey' and 'Mercada'.ch-chloroplast, etetioplast, etletioplast-like plastid, Tythylakoid.Chloroplasts that were observed only in cv.'Jersey' are characterised by well-developed grana, whereas etioplast contain prolamellar body.Etioplast-like plastid in cv.'Mercada' was more advanced in development and contained single perforated thylakoids and incipient grana without organized structure.

Figure S6 .
Figure S6.The relative expression level of genes related to plastid biogenesis, chloroplast differentiation and photosynthesis in albino regenerants of cv.'Mercada' compared to albino regenerants of cv.'Jersey'.a-c The relative expression level of genes involved in transcription (a), protein import to plastid (b) and translation (c).d The relative expression level of genes related to chloroplast differentiation.e The relative expression level of genes related to photosynthesis.Graphs show mean values of n ≥ 3 with SEM for relative expression level normalised to albino regenerants (GP) of cv.'Jersey'.An asterisk presents a value significantly different between green regenerants of 'Jersey' and 'Mercada' cultivars (t-Student test, P < 0.05).

Table S1 .
List of genes and primers used to perform RT-qPCR analysis

Table S3 .
List of genes, genome localisation and primers used to evaluate plastid DNA copy number using qPCR LSC -long single copy, IR -inverted repeat, SSC -short single copy.