Androgenic potential of ‘Jersey’ and ‘Mercada’ cultivars
Plant regeneration in isolated microspore culture of cvs. ‘Jersey’ and ‘Mercada’ occurred via process of microspore embryogenesis in which several phases could be distinguished. The first stage after pretreatment - induction of sporophytic development, involved cell divisions within the exine wall that led to the formation of a multicellular structure on the 7th day of culture (7dC, Fig. 1a). Rapture of the exine wall and further cell divisions led to the formation of structures that could be observed on the 21st day of culture (21dC) as globular pro-embryos (Fig. 1b). At this stage, the developed pro-embryos were transferred onto differentiation medium for two weeks (Fig. 1e). On the 35th day of culture (35dC), the meristematic zone could be recognised in differentiating embryos (Fig. 1c). Then, the embryos were transferred onto regeneration medium, for the first five days in dark and next in light (Fig. 1e). Already after eight days on regeneration medium, on the 43rd day of culture (43dC), the embryo body axis was detectable (Fig. 1d). The fully developed androgenic embryos, ready to convert into plants were visible on 46th day of in vitro culture (46dC, Fig. 2a). The further stages of plant regeneration presented in Fig. 2a,b refer to the time points used in this study for molecular analysis. It should be underlined that microspore-derived embryos of both cultivars reached the same stage of development at the same time.
Both cultivars selected for the study, ‘Jersey’ and ‘Mercada’, exhibited a high regeneration potential in isolated microspore culture and produced over 100 plants per 100,000 plated microspores. Along with the high overall number of regenerants, ‘Jersey’ and ‘Mercada’ differed in the frequency of regeneration of green plants. ‘Jersey’ regenerated 90% green plants whereas ‘Mercada’ produced only 5-10% green regenerants and the majority of regenerants were albino, irrespectively of the stress treatment applied for inducing of androgenesis (Fig. 2c). A slight increase in the contribution of green plants among regenerants was observed in ‘Mercada’ culture after SMB1 pre-treatment, but it was accompanied with reduction of the overall regeneration ability, compared to the cold pre-treatment (Fig. 2c). These results suggest that the modification of inductive treatment did not overcome the genotype-dependent formation of albino regenerants in barley androgenesis.
Plastid biogenesis and development during microspore-derived embryo formation and differentiation
At the stage of culture initiation, cvs. ‘Mercada’ and ‘Jersey’ showed differences in the types of plastids presented in the mid-to-late (ML) microspores utilised to initiate in vitro culture. TEM observation allowed to distinguish different types of plastids harboured by microspores and to estimate their numbers. The number of plastids was assessed per 25 µm2 of microspore cytoplasm and then per 25 µm2 of embryo cell cytoplasm in each cultivar. Cv. ‘Mercada’, that produced mostly albino plants in androgenesis, contained 50% amyloplasts that were filled with starch grains and 50% proplastids, whereas cv. ‘Jersey’ contained only initial and differentiating proplastids. The initial (undifferentiated) proplastids showed the single invaginations from the inner membrane and a low electron density, while the electron density of the differentiating proplastids was much higher . After pre-treatment, till 2dC, no changes occurred in the number and types of plastids presented in both cultivars. On 4dC, the proplastid differentiation into amyloplasts was observed in cv. ‘Jersey’ and the increase in the number of amyloplasts in cv. ‘Mercada’. At the end of pro-embryo formation, on 21dC, the number and types of plastids were similar in both cultivars, with ca. 60% of amyloplasts (Fig. 3a). Interestingly, at the end of differentiation phase, on 35dC, the number of plastids rapidly decreased in both cultivars, and initial proplastids represented 50% of observed plastids. The similar number and types of plastid were observed in apical domain of fully developed androgenic embryos of both cultivars (43dC) which implies that androgenic embryos contain the same categories of plastids, irrespectively of the types of produced regenerants.
Taking into account the presence of amyloplast in microspore-derived embryos of both cultivars, we analysed the expression of genes encoding isoforms of enzymes involved in reserve and assimilatory starch synthesis (Additional file 1: Table S1). At the stage of culture initiation, in ML microspores the expression level of genes Dpe2, GBSSI and Sbe1 involved in reserve starch synthesis was significantly higher in cv. ‘Mercada’, which was in accordance with the presence of amyloplasts observed exclusively in microspores of this cultivar (Fig. 3b). During isolated microspore culture, the expression of these genes decreased in both cultivars, which indicates the inhibition of reserve starch synthesis after stress treatment and induction of androgenesis. Contrary, the GBSSIb and SSIIb genes encoding enzymes of assimilatory starch synthesis increased gradually after pre-treatment in both cultivars (Fig. 3c). In cv. ‘Jersey’ the increase in expression of GBSSIb and SSIIb genes was observed already on 2dC and 4dC, respectively which explains the presence of starch-accumulating plastids observed in this cultivar on 4dC. The highest expression level of GBSSIb and SSIIb genes was observed on 21dC in developed pro-embryos of both cultivars. Interestingly, during embryo induction and pro-embryo development on 7dC and 21dC, no significant differences were observed between tested cultivars in the expression level of most genes related to plastid biogenesis that are involved in transcription and translation occurring in plastids. It should be noted that expression of these genes was higher in the cv. ‘Mercada’ compared to ‘Jersey’ at the stage of culture initiation (Fig. 4; Additional file 1: Figure S1) and expression of most of the genes involved in transcription including NEP (RpoTp gene), PEP subunits (encoded by genes rpoA, rpoB, rpoC1, rpoC2) was again higher in cv. ‘Mercada’ at the end of embryo differentiation and body axis formation (35dC and 43dC; Fig. 4). Such differences were not observed for the majority of genes involved in translation that encode plastid rRNA (Fig. 4c) and proteins of ribosome subunits (Additional file 1: Figure S1).
Taking into account these data, we conclude that green and albino producing cultivars did not show any substantial differences in plastid types and numbers during androgenic embryo induction and differentiation stage, however the developed embryos on 35dC and 43dC differed at the expression level of genes related to plastid biogenesis.
The fluctuation of plastome copy numbers during isolated microspore culture
Together with the plastid number, we assessed the number of plastid genomes during isolated microspore culture using qPCR. During culture initiation and embryo formation, the plastome copy number, which in ML microspores was twice as high in cv. ‘Jersey’ than ‘Mercada’, increased significantly in both cultivars and reached the highest value on 21dC (Fig. 5a). During differentiation of androgenic pro-embryos, between 21dC and 35dC, the rapid decrease (9 and 7 times in cv. ‘Jersey’ and ‘Mercada’, respectively) in plastome copy number was detected, parallel to the observed reduction in the number of plastids. However, while the number of plastids in the developed androgenic embryos on 43dC remained similar in both cultivars, the plastome copy number in plastids of cv. ‘Mercada’ was twice lower than in cv. ‘Jersey’ (Fig. 5a). Additionally, since the 35dC and the decline in the plastome copy number, the significant deviation of the copies of individual plastid genes was observed in cv. ‘Mercada’ (Fig. 5b).
The increase of the plastome copy number after pre-treatment indicated the induction of plastome replication process, therefore we analysed the expression profile of Polγ gene encoding the organellar DNA polymerase called also PolIA . Expression of Polγ gene was similar in ML microspores of both cultivars (Fig. 5c). During embryo induction phase, from 2dC to 7dC, the Polγ expression level in cv. ‘Mercada’ was much lower than in cv. ‘Jersey’ but equalized at the end of pro-embryo formation (21 dC, Fig. 5c). Expression of Polγ gene profiled similarly in both cultivars during embryo differentiation and body axis formation. The increased number of plastome copies observed since 46dC (Additional file 1: Figure S3) suggests that plastome replication was still active in both cultivars.
Since the culture is induced from immature pollen, we analysed the expression level of DPD1 gene encoding organelle exonuclease that acts during pollen development in vivo and degrades organellar DNA during plastid differentiation. At the stage of culture initiation, the expression level of DPD1 gene was 2-2.5 times higher in ML and pre-treated microspores of cv. ‘Mercada’ than cv. ‘Jersey’ and decreased during culture initiation (Fig. 5c). DPD1 gene was active in cv. ‘Mercada’ already in microspores at the early stage of development, before culture initiation (Additional file 1: Figure S2). This suggests an earlier activation of plastome degradation in cv. ‘Mercada’ during microspore development preceding culture initiation.
Differences in plastid biogenesis between cvs. ‘Jersey’ and ‘Mercada’ during regeneration of microspore-derived plants
Despite the similar average number of plastome copies in both cultivars at the end of body axis formation phase (43dC), the deviation in the copy number of particular plastid genes could possibly affect the further plastid development, therefore we continued the analysis of gene expression involved in plastid biogenesis (Additional file 1: Table S1) during regeneration of androgenic plants (46dC, 50dC and 55dC).
The average plastome copy number in cvs. ‘Jersey’ and ‘Mercada’ increased in converting embryos and regenerated plant compared to 43dC, which indicated the replication of plastid genomes, correlated with the increased expression of Polγ gene in cv. ‘Jersey’ (Additional file 1: Figure S3a,c). However, as we observed previously in. cv. ‘Mercada’ during embryo development, also during embryo conversion and regenerant development the copy number of particular genes did not represent the expected value regarding their localisation in the plastid genomes (Additional file 1: Figure S3b).
What was the most striking, during regeneration of cv. ‘Jersey’ plants, the expression of 16S and 23S genes encoding plastid rRNA increased immensely since 46dC (20 to 30-fold) to reach 300-500 times higher level on 55dC. In contrast to ‘Jersey’, during regeneration of cv. ‘Mercada’ plants, we did not observe any significant increase in the level of plastid-encoded rRNA transcripts between 46dC and 55dC (Fig. 6a). The expression of 16S and 23S genes in ‘Mercada’ albino regenerants on 55dC remained at the same level as in 43-day old embryos, which had only the visible body axis. The expression of other tested genes related to translation: rps8, rpl16 encoding proteins of small and large subunits of ribosome and infA, InfB encoding translation initiation factors increased in both cultivars during regeneration (Fig. 6b,c). Nonetheless, this growth was observed in cv. ‘Jersey’ already on 46dC, while in cv. ‘Mercada’ on 55dC in developed regenerants. On 55dC the level of expression of translation-related genes, except for InfB, was similar in both cultivars.
We showed that the vast increase in plastid rRNA transcript level was correlated with the regeneration of green plants during conversion of androgenic embryos. As a high level of expression of plastid rRNA genes is provided only by transcription carried by PEP (Plastid-encoded RNA polymerase), we analysed expression of other genes related to the process of transcription occurring in plastids. They were genes encoding proteins such as NEP (Nuclear-encoded RNA polymerase), subunits of PEP, SIG2 (sigma factor2 involved in transcription of plastid tRNAs) and trnE (tRNAGlu), all known to be involved in the switch between NEP- and PEP-dependent transcription [47, 49].
In androgenic embryos of cv. ‘Mercada’ the expression level of RpoTp gene encoding NEP was three times higher than in cv. ‘Jersey’ on 43dC. In the further stages of regeneration the expression of this gene was similar in both cultivars, with the highest level in cv. ‘Mercada’ on 55dC (Fig. 7a).Two plastid-localised genes rpoA and rpoB encoding subunits α and β of PEP showed diverged profiles of expression between cvs. ‘Jersey’ and ‘Mercada’. In cv. ‘Jersey’, expression of rpoA increased gradually during embryo conversion and reached the highest level on 55dC, while in ‘Mercada’ the increase in rpoA transcript level could be observed only on 55dC (Fig. 7b). It should be noted that the plastid-localised rpoA gene is transcribed preferentially in greening leaves by the PEP itself [42, 67], so a low level of its transcription indicated a low activity of PEP in regenerating ‘Mercada’ embryos. Contrary to the rpoA, the rpoB gene encoding the β subunit of PEP, is preferentially transcribed by the NEP . The transcription level of rpoB remained low and unchanged in ‘Jersey’ throughout the whole regeneration phase, while in ‘Mercada’ it increased significantly on 55dC compared to 43dC (Fig. 7b).
The high level of rpoB transcripts in ‘Mercada’ plants and differences in expression profiles of RpoTp and rpoA genes between both cultivars suggested that NEP was constantly active in plastids of regenerating ‘Mercada’ embryos and regenerated plants. Consequently, we performed analysis of transcription profile of tRNAGlu and Sig2 genes whose expression changes are related to transition from NEP to PEP-dependent transcription. Sig2, located in nucleus, encodes a sigma factor necessary for transcription initiation of plastome genes by PEP. Among these genes is tRNAGlu, whose transcription product, after reaching a certain level, inhibits the activity of NEP in plastids . Both genes: Sig2 and tRNAGlu, showed a similar expression pattern during regeneration but different for both cultivars. In cv. ‘Jersey’ the expression levels of Sig2 and tRNAGlu genes were significantly higher than in cv. ‘Mercada’ at each time point of plant regeneration (Fig. 7). Already on 46dC the expression levels of Sig2 and tRNAGlu genes were 37 and 5 times higher than on 43dC in cv. ‘Jersey’, while they remained unchanged until 55dC in cv. ‘Mercada’. At this time point a remarkably high increase of Sig2 and tRNAGlu expression was observed in ‘Jersey’ (380 times and 20 times higher compared to 43dC, respectively). These results indicate that the transcription occurring in plastids of ‘Jersey’ embryos was dependent on PEP as early as embryo conversion stage, whereas at the same developmental stage in ‘Mercada’ embryos, the transcription was still predominantly carried by NEP.
Chloroplast differentiation during regeneration of androgenic plants
The light-induced proplastid-to-chloroplast transition is a required step for further chloroplast differentiation that involves the efficient activation of expression of plastid genes, including rRNA genes, and induction of photomorphogenesis. Thus, during regeneration of androgenic plants we performed the analysis of expression of genes related to photomorphogenesis (genes encoding phytochromes and transcription factors that regulate photomorphogenesis), as well as genes involved in chloroplast differentiation, including regulation of chlorophyll synthesis, thylakoid synthesis and docking (Additional file 1: Table S1).
No significant differences in expression profiles of PhyA and PhyB encoding phytochromes were observed between cvs. ‘Jersey’ and ‘Mercada’ during regeneration, except for androgenic embryos on 43dC (Fig. 8a). We observed, however, starting from 46dC, substantial differences between both cultivars in expression of Glk1 and Glk2 genes encoding transcription factors that are positive regulators of photosynthesis-associated nuclear genes. In cv. ‘Jersey’ there was a significant increase of both genes expression, reaching 100-200 times higher level on 55dC compared to 43dC (Fig. 8b). Contrary to ‘Jersey’, in cv. ‘Mercada’ expression of both Glk genes was constant during embryo conversion and plant regeneration. The high increase of Glk genes expression in cv. ‘Jersey’, observed already in converting embryos, indicates the activation of photomorphogenesis in ‘Jersey’ plastids in the GLK-dependent pathway. The lack of increased expression of Glk genes in cv. ‘Mercada’ suggests that chloroplast differentiation was detained during regeneration stage in this genotype.
A significant difference was observed in expression profile of PIF1 regulating the chlorophyll synthesis which is also affected by light perception. In cv. ‘Jersey’ activation of PIF1 expression was observed in converting embryos on 46dC, whereas in cv. ‘Mercada’ a twice lower expression compared to cv. ‘Jersey’ was observed, that was stable throughout the whole regeneration period (Fig. 8c). Such differences between cultivars were not observed in expression profiles of Hy5 encoding another transcription factor involved in activation of nuclear genes controlling chloroplast development and PGP1 and RABA5e genes involved in synthesis and docking of thylakoid membrane, respectively, which are not affected by light perception (Additional file 1: Figure S4). PIF1 is a bHLH transcription factor, which is a critical modulator of cotyledon greening of dark-grown seedlings. PIF1 promotes seedling greening in two ways: first, it represses the accumulation of protochlorophyllide by regulating the expression of genes involved in the tetrapyrrole pathway; second, PIF1 directly binds to the promoter of PORC to activate its transcription, thus promoting the catalysis of protochlorophyllide into chlorophyll [68, 69].
Transmission electron microscopy analysis was performed to evaluate the number and describe types of plastids present in converting embryos and in leaves of regenerated plants on 43, 46, 50 and 55 day of culture of ‘Mercada’ and ‘Jersey’. No differences in plastid number per 25 µm2 of embryo cell cytoplasm between ‘Jersey’ and ‘Mercada’ were observed in the subsequent days of regeneration (Fig. 8d). Also, on 43dC, similar types of plastids (initial proplastids, proplastids and amyloplasts) were observed in similar proportion in both genotypes. However, since 46dC, when shoot apex become clearly visible in converting embryos, the significant differences were present in plastid types between analysed cultivars. In mesophyll cells of ‘Jersey’ regenerants, the majority of plastids were represented by chloroplasts, and only a small number of etioplasts was present on 46dC (Fig. 8d). Chloroplasts were characterised by well-developed grana, whereas etioplast contained only prolamellar body (Additional file 1: Figure S5). Later on, on 50dC and 55dC, only chloroplasts were observed in leaves of ‘Jersey’ regenerants, 90% of which were green. On the other hand, in cv. ‘Mercada’ that regenerated mostly albino plants, on 46dC a similar fraction (50%) of proplastids etioplast-like plastids was observed (Fig. 8d). Similar results were obtained for mesophyll cells of ‘Mercada’ regenerant leaves on 50dC and 55dC. The etioplast-like plastids in cv. ‘Mercada’ regenerants were more advanced in development and contained single perforated thylakoids and incipient grana without organized structure (Additional file 1: Figure S5).
Characterisation of albino regenerants of cvs. ‘Jersey’ and ‘Mercada’
Cv. ‘Jersey’, which in isolated microspore culture regenerated mostly green plants, produced also a small number (5-10%) of albino regenerants. To determine whether the processes that caused the albino formation in ‘Jersey’ were similar to those which occurred in ‘Mercada’, we compared expression profiles of genes involved in plastid biogenesis, chloroplast differentiation, photomorphogenesis and photosynthesis (Additional file 1: Table S1), as well as plastid ultrastructure and plastome replication in the albino and green regenerants of both genotypes.
The albino plants of both cultivars differed from the green regenerants of the corresponding cultivar in the expression profile of genes related to plastid transcription, translation and protein import to plastids (Fig. 9a). In albino regenerants of cv. ‘Jersey’ most of the tested genes engaged in these processes exhibited significantly lower expression compared to green regenerants. What is more, the relative expression levels of the genes that are associated with the transition from NEP to PEP transcription (rpoA, Sig2, tRNAGlu) were 2 to 5-fold lower, whereas the activity of genes encoding subunits of PEP (rpoB, rpoC1 and rpoC2), that are predominantly transcribed by NEP, were significantly higher than in green regenerants. This data suggested that transcription occurring in plastids of ‘Jersey’ albino plants was not switched to the PEP-dependent process and the NEP was the leading plastid RNA polymerase, similarly to the plant regeneration in cv. ’Mercada’ cultures.
Albino regenerants of cv. ’Jersey’ and cv. ‘Mercada’ exhibited a very low level of the plastid rRNA transcripts which was visualised by quality control of RNA samples of green and albino regenerants using Agilent 2100 Bioanalyzer (Fig. 9b). However, contrary to cv. ‘Jersey’, the albino plants of cv. ‘Mercada’ showed a similar or even higher expression level of the majority of analysed genes (except for tRNAGlu, Sig2, rRNAs) than green regenerants of this cultivar (Fig. 9a). These results indicate that transcription of genes involved in plastid biogenesis was still ongoing in albino plants of cv. ‘Mercada’. Furthermore, when the albino regenerants of two cultivars were compared, the relative expression level of the majority of genes related to plastid biogenesis in cv. ‘Mercada’ was higher than in cv. ‘Jersey’, independently from the gene localisation in the plastid or nuclear genome (Additional file 1: Figure S6). For example, RT-qPCR analysis revealed that the expression of 16S and 23S rRNA was two to four times lower in albino plants of cv. ‘Jersey’ than ‘Mercada’ (Additional file 1: Figure S6).
Albino regenerants of both, ‘Jersey’ and ‘Mercada’ exhibited a significantly lower, than green plants, expression of genes encoding GLK transcription factors participating in regulation of photomorphogenesis. Also the transcription activity of other genes involved in chloroplast differentiation, such as PIF1 (regulating the chlorophyll synthesis), PGP1 (involved in synthesis of thylakoid membrane) and RABA5e (responsible for docking of thylakoid membrane) was reduced in albino regenerants of cv. ‘Jersey’ (Fig. 10). Interestingly, in albino regenerants of cv. Mercada’, the transcription level of all examined genes related to photomorphogenesis was similar or higher compared to the green plants. These results were in agreement with the observations of plastid ultrastructure in regenerants of both genotypes. TEM analysis of plastids present in leaf mesophyll cells of albino regenerants revealed differences between ‘Jersey’ and ‘Mercada’. In albino regenerants of both cultivars, we identified only differentiating proplastids and plastid similar to etioplasts, however at different stages of differentiation (Fig. 9c,d). That etioplast-like plastids in ‘Jersey’ contained only the prolamellar body, and no structures similar to prothylakoids were observed (Fig. 9d). Whereas in the case of etioplast-like plastids in ‘Mercada’, both the prolamellar body and non-organized prothylakoid/thylakoid structures were present (Fig. 9d). Since in both genotypes the conversion of observed plastids to chloroplast did not occurred, these plastids cannot be described as functional etioplasts. Contrary to albino plants, the structure of chloroplasts present in green regenerants did not differ between cultivars studied (Fig. 9d).
As a result of the failed plastid-to-chloroplast transition, albino regenerants of both, ‘Jersey’ and ‘Mercada’ exhibited significantly lower expression of plastid and nuclear genes that encode subunits of photosystems I and II, ATP synthase, cytochrome f, NADH-PQ oxidoreductase and RubisCo complex, compared to the green regenerants of a corresponding cultivar (Fig. 10).
In addition to gene expression and plastid ultrastructure, we assessed the copy number of plastome-localised genes in green and albino regenerants. In green plants of both cultivars, the number copies of individual genes was consistent with their localisation within the plastome. On the contrary, the albino plants of both cultivars contained various number of copies of individual genes, which were inconsistent with their predicted number based on gene location in the plastid genome (Fig. 11). In ‘Jersey’, the average plastome copy number was three times lower in albino plants, compared to green regenerants (Fig. 11a). Additionally, genes localised in inverted repeats of the plastid genome did not occur in the predicted double number of copies compared to genes located in single copy regions. This suggests that number of complete plastomes and thus templates were limited in albino plants of cv. ‘Jersey’, which in turn might result in the low expression level of plastidial rRNA genes, the arrest of chloroplast differentiation and albino phenotype of regenerants carrying these changes.
Contrary to ‘Jersey’, in albino regenerants of cv. ‘Mercada’ the average number of plastomes was two times higher than in green plants (Fig. 11b). As we observed already during plant regeneration (Additional file 1: Figure S3b), the copy number of individual genes in ‘Mercada’ albino plants was not consistent with the value predicted on the basis of gene localisation. What was interesting, the genes psbA and matK localised proximal to the replication initiation site showed even three times higher number of copies in albino compared to green regenerants (Fig. 11b). The copy number of genes decreased along with the distance from the replication initiation site, which may indicate that replication has been often initiated, but not completed.
Additional support for this observation was provided by expression analysis of Polγ gene whose expression was two-fold higher in the albino than in green regenerants of cv. ‘Mercada’. The opposite expression pattern was observed for cv. ‘Jersey’, in which the expression of Polγ was two times higher in green than albino plants (Fig. 11c).
Comparison of cvs. ‘Jersey’ and ‘Mercada’ clearly showed that the cultivars presenting opposing ratio of green and albino regenerants showed the arrest of chloroplast differentiation at an early stage of biogenesis. This arrest was related to the lack of PEP-dependent transcription and inhibition of photomorphogenesis.