Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin.

Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos.

Introduction

Found in all nucleated organisms, hemoglobins are important Fe heme-containing proteins fulfilling a variety of tasks. Initially characterized in vertebrates in relation to their ability to bind and transport oxygen and other ligands such as CO2, and NO, hemoglobin-like compounds were also found expressed in the nodules of legumes containing nitrogen-fixing bacteria where they served to prevent inactivation of nitrogenase by binding oxygen (reviewed in Smagghe ). Additional plant hemoglobin-like coumpounds were subsequently found to be more widely distributed in plants (Hill, 2012) and were ascribed the name, nonsymbiotic hemoglobins to distinguish them from leghemoglobin. The types (Garrocho-Villegas ) and function (Hill, 2014) of nonsymbiotic hemoglobins have expanded considerably to the point that a more specific name, phytoglobin (Pgb), has been introduced.

From a phylogenetic perspective, oxygen binding activity, and the expression profile, three types of plant Pgbs have been described (Hunt ). Class 1 and 2 Pgbs possess a 3-on-3 α-helical loop surrounding the heme moiety, while class 3 Pgbs are similar to truncated bacterial globins (Watts ). The majority of the studies are centered on class 1 and 2 which are characterized by a very high oxygen binding affinity: an approximate K m of 2nM for members of class 1 and 150nM for members of class 2 (Hoy and Hargrove, 2008; Dordas, 2009). The high affinity for oxygen exhibited by class 1 Pgbs discounts their roles in oxygen sensing and transport (Hill, 1998). The most plausible function of plant Pgbs is to scavenge NO, as demonstrated in several developmental and stress responses (reviewed in Hill, 2012).

Three Pgb genes categorized in their respective classes have been identified in Arabidopsis: PGB1, PGB2, and PGB3. While no information is available on the function of PGB3, several studies have provided evidence for the role of PGB1 and PGB2 during hypoxia. Due to its ability to scavenge NO efficiently under hypoxic conditions, PGB1 exercises a protective role during abiotic stress (Perazzolli ). Arabidopsis roots grown under low oxygen conditions rapidly expressed PGB1, and plants overexpressing PGB1 exhibited a higher survival rate resulting from a depletion in cellular NO (Hunt ). The role of protection from hypoxia observed for PGB1 was also documented with other class 1 Pgbs. Hypoxic roots of alfalfa overexpressing a class 1 Pgb developed fewer aerenchyma and exhibited enhanced growth due to sustained NO-scavenging mechanisms (Dordas ). Opposite results were observed in plants suppressing the barley Pgb (Dordas ).

Like PGB1, overexpression of PGB2 increased NO scavenging (Hebelstrup and Jensen, 2008; Hebelstrup ) and enhanced plant survival under hypoxic conditions (Hebelstrup ). Expression of PGB2 is favored by cytokinin and low temperatures (Trevaskis ; Hunt ), conditions not affecting PGB1 and denoting differential control mechanisms and possibly functions of the two Pgbs. The preferential expression of PGB2 in immature and developing organs, such as somatic embryos, leaflets, and immature seeds and fruits (Hendriks ; Hunt ; Wang ), is suggestive of a function for the gene to accommodate the high energy demand of the organs. In developing Arabidopsis seeds, overexpression of PGB2 has been associated with improved oil accumulation through the maintenance of a high energy status (Vigeolas ).

A recurring theme emerging from these studies is the mediation of NO in many Pgb-regulated events. The prominent role of NO as a signal molecule in many physiological responses, in conjunction with the expression of Pgbs under normoxic conditions (Hill, 2012), suggests a possible involvement of Pgbs in fundamental developmental processes. Major phenotypic defects were observed in Arabidopsis plants with altered PGB1 and PGB2 expression (Hebelstrup ). Independent evidence suggests a control of meristem function by Pgbs. While overexpression of either PGB1 or PGB2 encourages the vegetative–reproductive transition of the shoot meristem, the repression of PGB1 affects the time of flowering (Hebelstrup and Jensen, 2008). Meristem formation in vitro was also affected by Pgbs, with the overexpression of both PGB1 and PGB2 favoring the formation of shoots through the activation of auxin and cytokinin perception (Wang ). By modulating NO emission, PGB1 and PGB2 also regulate hyponastic responses during flooding, an observation integrating Pgbs in long-range plant signaling mechanisms (Hebelstrup ).

While the participation of Pgbs in developmental processes has mainly been investigated post-embryonically, Pgbs might play a central role during plant embryogenesis. Phytoglobin genes are expressed during embryo development (Smagghe ), and when hemoglobin is applied exogenously it influences somatic embryogenesis, the ability of somatic cells to produce embryos (Jayabalan ). A more direct involvement of Pgbs during embryo formation was demonstrated by Elhiti using Arabidopsis somatic embryogenesis. Suppression of PGB2 increased embryogenesis by elevating NO levels at the sites of the explants forming somatic embryos. Accumulation of NO suppresses MYC2 (Elhiti ), a basic helix–loop–helix (bHLH) domain-containing transcription factor which represses the biosynthesis of auxin (Dombrecht ), the inductive signal which initiates the embryogenic process (Raghavan, 2004). As a result of this mechanism, PGB2-suppressed cells accumulate more auxin and produce a large number of somatic embryos (Elhiti ). While representing a valid framework integrating Pgb signaling in plant embryogenesis, this model is most probably incomplete in terms of the number of intermediates transducing the PGB2 response. During post-embryonic growth, both NO and MYC2 operate at the interphase of a variety of transduction pathways often involving hormones, predominantly jasmonic acid (JA) (Chen ; Mur ). While the link between MYC2 and JA signaling has been well established during plant–pathogen interactions and insect predation (Lorenzo and Solano, 2005), the relationship between NO and JA is far from clear. JA synthesis is repressed by NO in some systems but induced in others (Orozco-Cardenas and Ryan, 2002). A rapid induction in JA level following inoculation with Botryts cinerea was observed in Arabidopsis plants accumulating NO through suppression of Pgb (Hebelstrup ). Based on these observations, it cannot be excluded that JA plays a key role during embryogenesis, possibly as an integrated component of the PGB2 regulatory mechanisms.

In an effort to establish a relationship between PGB2, NO, and JA in the regulation of embryo formation, we used the well-characterized Arabidopsis somatic embryogenesis system (Elhiti ). Formation of somatic embryos in Arabidopsis is a two-step process (Fig. 1A). The first involves culturing early cotyledonary zygotic embryos on an auxin-containing induction medium which stimulates the formation of embryogenic tissue. Production of somatic embryos from the embryogenic tissue is then initiated by the removal of auxin (Bassuner ). Our results suggest that JA is a key component of PGB2 regulation of embryogenesis in a model including NO and several JA-responsive intermediates.

Fig. 1.

Effects of altered levels of jasmonic acid (JA) on Arabidopsis somatic embryogenesis. (A) Arabidopsis somatic embryos are generated through a two-step process. Dissected zygotic embryos are initially plated on 2,4-D-containing induction medium required for the formation of embryogenic tissue. After 14 d, the explants are transferred onto a hormone-free development medium which stimulates the production of somatic embryos. Fully developed somatic embryos can be obtained after 9 d on development medium. Arrows indicate the formation of the embryogenic tissue. (B) Changes in embryo number in the wild type (WT) line cultured with increasing levels of JA applied to the induction medium. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the control (JA=0nM) value set at 100%. (C) Effects of applications of JA (50nM) and/or Phenidone (Ph) on the number of somatic embryos produced by the WT, pgb2, and 35S:PGB2/pgb2 lines. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from value of the WT (C) set at 100%. (This figure is available in colour at JXB online.)

Materials and methods

Plant materials

The Arabidopsis (Columbia) mutant lines ami1-1 (SALK_069970) (Elhiti ), jaz1-1 (SALK 011957) (Demianski ), and aos1-1 (SALK 017756) (Park ), and the pASA1:GUS reporter line (CS16701), were obtained from the Arabidopsis Biological Resource Center (ABRC). The following lines were received as gifts: the pgb2 knock-out line (referred to as glb2 in Hebelstrup ); the myc2-1 mutant and the 35S:MYC2 line (Dombrecht ); the 35S:JAZ1 line (Thines ); the pJAZ1:GUS-GFP line (Gutierrez ); the 35S:MYC2-GUS line (Zhai et al., 2013); the pYUC4:GUS line (Eklund ); and the pPDF1.2:GUS line (Koorneef ). pgb2-aos1-1 double mutant lines were generated by crossing (Supplementary Fig. S1 at JXB online).

Growth conditions and induction of somatic embryogenesis

Arabidopsis seeds were sterilized (70% ethanol+0.5% Triton X-100 for 15min followed by 95% ethanol for 15min) and plated on germination medium (half-strength MS; Murashige and Skoog, 1962). The plates were kept at 4 °C in the dark for 2–3 d and then transferred to a growth cabinet (20–22 °C, 16h light/8h dark photoperiod). Plants were grown until siliques were formed, ~21–28 d.

Somatic embryogenesis was promoted using a modified method based on that described by Bassuner . Immature zygotic embryos were plated on induction medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) for 14 d, followed by transfer onto hormone-free development medium. Fully developed somatic embryos were counted after 9 d.

Chemical treatments

The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and the NO donor sodium nitroprusside (SNP) were applied as specified in Elhiti . Applications were performed by dispensing 10 μl of a 10 µM solution directly on the explants every other day throughout culture in the induction medium.

JA (Sigma) was dissolved in water and added to the culture medium at different concentrations as reported in the text. The JA inhibitor 1-phenyl-3-pyrazolidinone (Phenidone, Ph) was applied at a concentration of 10nM.

Total RNA isolation and quantitative real-time PCR analysis

Total RNA was extracted with TRIzol reagent (Invitrogen), treated with DNase I (RNase-free, Promega), and utilized for cDNA synthesis with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

Quantitative real-time PCR was performed as described in Elhiti using the primers listed in Supplementary Table S1. The relative level of gene expression was analyzed with the 2−∆∆ method described by Livak and Schmittgen (2001) using UBQ10 (AT4G05320) as a reference (Czechowski ; Hong ).

β-Glucuronidase assays

β-Glucuronidase (GUS) histochemical staining assay was performed as described by Sieburth and Meyerowitz (1997). The somatic embryos were examined and photographed using a dissecting microscope equipped with a Leica DC500 digital camera. A minimum of 20 samples per treatment were imaged.

IAA and JA immunolocalization

Immunolocalization of endogenous indole acetic acid (IAA) was carried out following the procedure used by Elhiti . Immunolocalization of endogenous JA was performed as described by Mielke , with some minor modifications. The plant material was fixed in 4% (w/v) 1-ethyl-3-(3–dimethylaminopropyl) carbodiimide (EDC) in phosphate-buffered saline (PBS) for 3h at room temperature. After dehydration in a graded ethanol series, the specimens were infiltrated in PEG-8 distearate containing low melting point wax (Electron Microscopy Sciences) at 45 °C. Sections (10 μm thickness) were incubated with anti-JA antibodies raised in rabbit (kindly donated by Professor House, IPK, Germany) diluted 1:1000 in PBS containing 5% (w/v) BSA and 1% (v/v) acetylated BSA (BSAac; Promega). The secondary goat anti-rabbit IgG antibody conjugated with AlexaFluor594 (Invitrogen) was used according to the manufacturer’s instructions at a 1:2000 dilution. Sections were analyzed by epifluorescence microscopy.

Statistical analysis

All experiments were performed using at least three biological replicates, and Tukey’s post-hoc test for multiple variance was used to compare differences among samples (Zar, 1999) (P=0.05) by the SPSS 14 statistical program.

Results

Repression of PGB2 increases embryogenesis through the NO-mediated elevation in JA level

Arabidopsis somatic embryogenesis is a two-step process (Fig. 1A). Dissected zygotic embryos were cultured on a 2,4-D solid induction medium for 14 d. After 3 d, the cotyledons of the explants started swelling and embryogenic tissue became apparent at day 7. During the following days on induction medium (days 7–14), the embryogenic tissue increased in size. Embryo production was stimulated by transferring the tissue onto a hormone-free development medium. As also reported previously (Elhiti ), the term somatic embryos used in this study refers to fully developed embryos collected after 9 d on the development medium (Fig. 1A).

In wild-type (WT) tissue, inclusion of JA in the induction medium affected the number of somatic embryos in a dose-dependent fashion, with the most pronounced increase observed with 50nM JA (Fig. 1B). This concentration was used to compare the effects of JA manipulations on somatic embryos produced by the WT line and the Pgb2 knock-out (pgb2) line characterized by a superior embryogenic performance (Elhiti ). In the WT, the JA stimulation of embryogenesis was reversed by Ph, a JA biosynthetic inhibitor (Farmer ; Bruinsma ), which strongly repressed embryo production when applied alone (Fig. 1C). The beneficial effect of JA on somatic embryogenesis was not observed in the pgb2 line. To prove that the increased number of embryos observed in the pgb2 line relative to the WT line was solely due to suppression of the gene, we overexpressed PGB2 in the mutant line. The resulting 35S:PGB2/pgb2 (Supplementary Fig. S2) had an embryonic yield comparable with the WT line (Fig. 1C)

The different embryonic behavior of the two lines following JA treatments was further examined in light of the following premises: PGB2 is an effective scavenger of NO (Hebelstrup ) expressed at the sites of the Arabidopsis explants producing embryogenic tissue (Elhiti ); NO accumulates preferentially in cells suppressing PGB2, and this accumulation is required for the enhanced embryogenic performance of the pgb2 line (Elhiti ).

To establish an experimental baseline for our experiments, we confirmed previous results related to the effects of NO manipulations on somatic embryogenesis (Elhiti ). Applications of the NO donor SNP increased embryo production in the WT line, whereas inclusion of the NO scavenger cPTIO compromised the embryogenic process in both WT and pgb2 lines (Fig. 2A). Compared with the WT, the number of somatic embryos more than doubled in the pgb2 line, an observation consistent with the higher level of NO accumulating in the latter (Elhiti ). However, the beneficial effects of high NO levels (either by SNP or by suppression of PGB2) on somatic embryogenesis were JA dependent. In the WT line, depletion of JA by Ph in an NO-enriched environment (SNP+Ph) suppressed embryo production, whereas elevated JA levels promoted embryogenesis even in an environment depleted of NO (cPTIO+JA) (Fig. 2A). Similar results were also observed in the NO-accumulator pgb2 line where applications of JA reversed the inhibitory effect of cPTIO (Fig. 2A). Therefore, the beneficial effects of high NO levels (by SNP or suppression of PGB2) on embryogenesis are mediated by JA.

Fig. 2.

The effects of nitric oxide (NO) on somatic embryogenesis are mediated by jasmonic acid (JA). (A) Effects of applications of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), the NO-releasing agent sodium nitroprusside (SNP), JA, and Phenidone (Ph) on the number of somatic embryos produced by the WT and pgb2 lines. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from value of the WT (C) set at 100%. (B) Immunolocalization of JA on the embryogenic tissue arising from the cotyledons of the explants after 7 d on induction medium. Tissue was treated with SNP, cPTIO, JA, and/or Ph. Primary antibodies were omitted from the negative control. Scale bars=20 μm.

We further assessed if JA accumulated preferentially in cells with increasing levels of NO. While JA quantitation cannot be performed in our system due to the reduced size of the embryos, immunolocalization of JA was conducted across the cotyledons of the explants. These are the regions generating the embryogenic tissue (Fig. 1A) and accumulating NO following suppression of PGB2 (fig. 3A in Elhiti ). An intense JA signal was observed in NO-enriched environments (WT tissue treated with SNP and pgb2 tissue) (Fig. 2B), while an experimental reduction in NO level by cPTIO reduced the fluorescence. Low JA signal was also observed in the 35S:PGB2/pgb2 line. Specificity of the antibody was verified using JA and/or Ph (Fig. 2B).

Collectively these results suggest that NO induces the accumulation of JA, and heightened levels of JA favor embryogenesis in a dose–response fashion, with exogenous applications increasing embryo number only in systems with reduced levels of endogenous JA (WT line), but not in others already enriched in JA (pgb2 line).

NO activates the expression of genes participating in JA biosynthesis

De novo biosynthesis of JA requires the co-ordinated expression of several genes including LYPOXYGENASE2 (LOX2) involved in the oxygenation of fatty acids to their hydroperoxy derivatives and ALLENE OXIDE SYNTHASE (AOS), forming unstable allene epoxides from the dehydratation of 1,3-hydroperoxy-octatrienoic acid (Wasterack and Hause, 2013). On day 3 and day 7 on induction medium (coinciding with the formation of embryogenic tissue Fig. 1A), the expression of both genes increased in NO-enriched environments (i.e. SNP-treated WT tissue and pgb2 tissue). Consistent with these observations, a reduction of NO level by cPTIO in both WT and pgb2 lines repressed the expression of both genes (Fig. 3A). Reintroduction of PGB2 in the pgb2 line reduced the expression levels of both LOX2 and AOS to WT values (Supplementary Fig. S3)

Fig. 3.

The effects of nitric oxide (NO) on jasmonic acid (JA) synthesis. (A) Expression level by quantitative (q)RT–PCR of the two JA biosynthetic genes [LIPOXYGENASE2 (LOX2) and ALLENE OXIDE SYNTHASE (AOS)] at days 3 and 7 in the induction medium of somatic embryogenesis. WT and pgb2 tissue was cultured in medium with altered levels of NO and JA using the pharmacological treatments described in Fig. 2. Values are means ±SE of at least three biological replicates and normalized to the value of the WT (C) of day 3 set at 1. Asterisks indicate statistically significant differences (P≤0.005) from the WT (C) value of the respective day in culture. (B) Number of somatic embryos generated from WT and aos plants. Explants were cultured on medium with altered levels of NO and JA. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the WT (C) value set at 100%. (C) Number of somatic embryos generated from WT, pgb2 aos, and pgb2/aos double mutant plants. Explants were cultured on medium with altered levels of NO and JA. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the WT value set at 100%.

The requirement for JA, and more specifically the expression of its biosynthetic gene AOS, as downstream components of the PGB2 and NO regulation of embryogenesis was further examined by analyzing the behavior in culture of the JA-deficient aos1-1 mutant (Park ), and the pgb2-aos double mutant (Fig. 3B, C). Somatic embryo production was significantly reduced by the suppression of AOS independently from NO levels and PGB2 expression. This reduction was partially reversed by exogenous JA (Fig. 3B, C).

Evidence presented here suggests that NO is a transcriptional activator of JA biosynthesis, and this activation is required for the PGB2 regulation of somatic embryogenesis.

Regulation of embryogenesis by JA is mediated by JAZ1 and MYC2

JAZ1 is a JA-inducible nuclear-localized protein belonging to the larger family of the TIFY proteins (Vanholme ) integrated in several JA biotic stress responses (Niu ; Sasaki-Sekimoto ). During somatic embryogenesis, JAZ1 expression is induced after 12h of JA application (Supplementary Fig. S4). On both day 3 and day 7 on induction medium, its expression increased in the pgb2 line and in response to pharmacological treatments elevating NO or JA levels, while it decreased following depletion of NO (by cPTIO) (Fig. 4A). Reintroduction of PGB2 in the pgb2 line reduced the expression levels of JAZ1 to WT values (Supplementary Fig. S3)

Fig. 4.

Nitric oxide (NO) and jasmonic acid (JA) affect JAZ1. (A) Expression level by quantitative (q)RT–PCR of JAZ1 at days 3 and 7 in the induction medium of somatic embryogenesis. WT and pgb2 tissue was cultured in medium with altered levels of NO and JA using the pharmacological treatments described in Fig. 2. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT (C) of day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT (C) value of the respective day in culture. (B) Localization patterns of JAZ1 by GUS staining at day 7 on induction medium. Tissue was subjected to the indicated pharmacological treatments. (C) Expression level by qRT–CR of JAZ1 in the WT and aos lines at days 3 and 7 on induction medium. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT at day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT value of the respective day in culture. (D) Number of somatic embryos generated from WT, 35S:JAZ1, and jaz1-1 plants. Explants were cultured on media with altered levels of NO and JA. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the WT value set at 100%.

The regulation of JAZ1 during the induction of embryogenesis was also confirmed using the pJAZ1:GUS reporter line showing a staining pattern in the apical regions of the explants, including the embryogenic tissue, mediated by NO and JA levels (Fig. 4B).

To verify further the requirement for JA for JAZ1 expression during somatic embryogenesis, we used the JA-deficient aos1-1 line. Compared with the WT, the expression of JAZ1 was significantly repressed in the aos1-1 explants on both day 3 and day 7 on induction medium (Fig. 4C).

Modulations in JAZ1 expression have profound effects on somatic embryogenesis. While the constitutive expression of this gene in the 35S:JAZ1 line increased embryo production, its suppression in the jaz1-1 mutant repressed embryogenesis (Fig. 4D). The fact that this repression was not rescued by manipulating the levels of NO (by SNP or cPTIO) or JA (by Ph or JA) suggests that JAZ1 is a downstream component in the regulation of embryogenesis.

Another key executor of the JA response during biotic and abiotic stress responses is MYC2, a bHLH domain-containing transcription factor (Dombrecht ). Expression of MYC2 shows a biphasic pattern in response to JA. While initial applications of JA increase MYC2 expression, prolonged exposure to JA repressed MYC2 expression (Supplementary Fig. S4).

MYC2 is repressed in PGB2-suppressed cells, and this repression promotes auxin production and favors somatic embryogenesis (Elhiti ). On both day 3 and day 7 on induction medium, MYC2 was down-regulated in high JA and NO environments (JA- or SNP-treated tissue and pgb2 tissue), while it was induced by cPTIO (Fig. 5A), or by the the overexpression of PGB2 in the pgb2 line (Supplementary Fig. S3). A significant induction of this gene also occurred in the JA-deficient aos1-1 explants on the same days in culture (Fig. 5B). Suppression in MYC2 in the myc2-1 mutant increased embryogenesis in a pattern consistent with previous studies (Elhiti ). This increase, however, was not affected by pharmacological treatments altering the levels of NO or JA (Fig. 5C).

Fig. 5.

Nitric oxide (NO) and jasmonic acid (JA) affect MYC2. (A) Expression level by quantitative (q)RT–PCR of MYC2 at days 3 and 7 in the medium with altered levels of NO and JA using the pharmacological treatments described in Fig. 2. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT (C) of day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT (C) value of the respective day in culture. (B) Expression level by qRT–PCR of MYC2 in WT and aos lines at days 3 and 7 on induction medium. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT at day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT value of the respective day in culture. (C) Number of somatic embryos generated from WT, 35S:MYC2, and myc2-1 plants. Explants were cultured on media with altered levels of NO and JA. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the WT value set at 100%.

These results suggest that transcriptional regulation of MYC2, like JAZ1, plays an important role in the NO and JA control of Arabidopsis somatic embryogenesis, and that increased embryo number is favored by the up-regulation of JAZ1 and the repression of MYC2.

The regulation of JAZ1 and MYC2 during the JA response in plant–pathogen interaction is complicated by feedback loops influencing the expression of both (Chini ; Thines ; Chung ; Kazan and Manners, 2013). To verify the presence of transcriptional regulatory mechanisms operating during somatic embryogenesis, the expression of the two genes was measured in lines suppressing or ectopically expressing MYC2 and JAZ1. On both day 3 and day 7 on induction medium, the expression of MYC2 was induced in the jaz1-1 line while it was repressed in the 35S:JAZ1 line. This pattern was in contrast to that observed for JAZ1 which was down-regulated in the myc2-1 line and up-regulated in the 35S:MYC2 line (Fig. 6).

Fig. 6.

Transcriptional regulation of MYC2 and JAZ1. (A) Expression level by quantitative (q)RT–PCR of MYC2 at days 3 and 7 in the induction medium of somatic embryogenesis in the WT, jaz1-1, and 35S:JAZ1 lines. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT of day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT value of the respective day in culture. (B) Expression level by qRT–PCR of JAZ1 at days 3 and 7 in the induction medium of somatic embryogenesis in the WT, myc2-1, and 35S:MYC2 lines. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT of day 3 set at 1. An asterisk indicates statistically significant differences (P≤0.005) from the WT value of the respective day in culture.

JA response modulates embryogenesis by altering IAA biosynthetic genes

During the initial phases of somatic embryogenesis, auxin is the inductive signal promoting the re-differentiation of the somatic cells and formation of embryogenic tissue (Raghavan, 2004). Previous studies showed that suppression of PGB2 increases auxin levels in a process dependent on the concurrent suppression of MYC2, an inhibitor of IAA synthesis (Elhiti ). To expand this model further, we assessed whether IAA biosynthesis was regulated by the JA response during embryogenesis. Production of IAA from indole-3-acetamide is modulated by AMIDASE1 (AMI1) (Pollman ). On both days 3 and 7 on induction medium, AMI1 expression was up-regulated by all those treatments increasing NO (SNP or suppression of PGB2) or JA, while it was suppressed by low levels of NO (cPTIO) or JA (Ph) (Fig. 7A). In addition, a depletion of JA in an enriched NO environment (SNP+Ph) decreased the expression of this gene while an enrichment in JA level under low NO conditions (cPTIO+JA) had opposite effects (Fig. 7A). Reintroduction of PGB2 into the pgb2 line reduced AMI1 expression to WT values (Supplementary Fig. S3). The JA requirement for the sustained expression of AMI1 was also confirmed using the JA-deficient aos1-1 line in which AMI1 transcript levels were reduced on both day 3 and day 7 on induction medium (Fig. 7B). To assess further if AMI1 regulation was downstream of the JA response, transcription studies were conducted in lines with altered expression of JAZ1 and MYC2. The transcript levels of AMI1 were induced by suppression of MYC2 (myc2-1 mutant) or ectopic expression of JAZ1 (35S:JAZ1) (Fig. 7C). Besides AMI1, two other IAA biosynthetic genes: ANTHRANILATE SYNTHASE-α SUBUNIT (ASA1), encoding a component of the early enzyme converting chorismate to ribosyl anthralilate, and YUCCA (YUC4) converting indole-3-yl pyruvate to (indol-3-yl)acetate (Cheng ; Zhao, 2011), show a similar JA-mediated transcriptional regulation (Supplementary Figs S3, S5, S6). The expression of both genes occurs mainly in the cotyledonary regions of the explant originating the embryogenic tissue (Supplementary Figs S5, S6).

Fig. 7.

Effects of jasmonic acid (JA) and nitric oxide (NO) on AMI1 expression. (A) Expression level by quantitative (q)RT–PCR of AMI1 at days 3 and 7 in the induction medium of somatic embryogenesis. WT and pgb2 tissue was cultured in medium with altered levels of NO and JA using the pharmacological treatments described in Fig. 2. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT (C) of day 3 set at 1. An asterisk indicates statistically significant differences (P<0.005) from the WT (C) value of the respective day in culture. (B) Expression level by qRT–PCR of AMI1 in WT and aos lines at days 3 and 7 on induction medium. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT of day 3 set at 1. An asterisk indicates statistically significant differences (P<0.005) from the WT value of the respective day in culture. (C) Expression level by qRT–PCR of AMI1 at days 3 and 7 in the induction medium of somatic embryogenesis in the WT, myc2-1, jaz1-1, and 35S:JAZ1 lines. Values are means ±SE of at least three biological replicates and are normalized to the value of the WT of day 3 set at 1. An asterisk indicates statistically significant differences (P<0.005) from the WT value of the respective day in culture.

Immunolocalization of the auxin IAA in the embryogenic tissue arising from the cotyledons of the zygotic explants confirmed the localization pattern of the biosynthetic genes (Fig. 8A). Tissues with elevated JA levels (WT+JA or pgb2) or subjected to conditions enhancing the JA response (myc2-1 mutant or 35S:JAZ1) had strong IAA signals. A reduction in IAA level occurred either in tissue depleted in JA level (pgb2 +Ph, aos1-1, pgb2/aos1-1), following conditions repressing the JA response (jaz1-1 and 35S:MYC2), or by the reintroduction of PGB2 in the pgb2 line (Fig. 8A). The requirement for IAA production in the JA regulation of somatic embryogenesis was further verified using the ami1-1 mutant, which significantly limits the beneficial effect of JA on embryo number (Fig. 8B).

Fig. 8.

Auxin and somatic embryogenesis. (A) Immunolocalization of IAA along the cotyledons of explants collected at day 7 on induction medium. Treatments described in Figs 6 and 7 were utilized. Primary antibody was omitted from the negative control section. (B) Number of somatic embryos generated from the WT and the ami1-1 mutant treated with JA. Values are means ±SE of at least three biological replicates. An asterisk indicates statistically significant differences (P≤0.005) from the WT value set at 100%.

Collectively, these data suggest that increased NO levels, resulting either from pharmacological treatments or by suppression of PGB2, increase JA production through the transcription of two key biosynthetic enzymes, LOX2 and AOS, which activate the JA response (Fig. 9). Key events in this response are the induction of JAZ1 and the repression of MYC2 which promote the accumulation of IAA within the embryogenic tissue and the formation of somatic embryos.

Fig. 9.

Suggested model describing the interaction among PGB2, NO, JA, and IAA during the induction phase of somatic embryogenesis in Arabidopsis.

Discussion

Generation of embryos in vitro is rendered possible by the inherent ability of plant cells to embark on novel developmental programs when subjected to specific changes in environmental conditions. This concept is best exemplified in Arabidopsis, where isolated zygotic embryos cultured on induction medium produce embryogenic tissue from the cotyledons; an event which becomes apparent after 3–7 d. The process is stimulated by the auxin 2,4-D, the inductive signal responsible for the de-differentiation of the cotyledon cells and proliferation of embryogenic cells (Raghavan, 2004). The embryogenic tissue is composed of immature embryos which are allowed to grow further on a development medium devoid of auxin (Fig. 1A). Production of somatic embryos in Arabidopsis is facilitated by the suppression of the phytoglobin PGB2, an effective NO scavenger (Hebelstrup ). Explants suppressing PGB2 accumulate NO within the embryogenic tissue and this promotes the formation of somatic embryos (Elhiti ). This enhanced embryogenic ability was solely due to the suppression of PGB2, as the reintroduction of PGB2 in the pgb2 line decreased the number of embryos to WT values (Fig. 1C)

In plants, NO participates in diverse physiological functions and operates at the interphase of many hormone-mediated responses (Neill ; Wilson ; Hancock ; Freschi, 2013; Simontacchi ). Involved in many developmental responses ranging from leaf expansion, root growth, and senescence (Wendehenne ; Yadav ), NO is tightly linked to JA in wounding and plant pathogen interaction (Grun ; Wang ). Having emerged as a downstream modulator in the JA response, NO has also been shown to contribute to JA synthesis (Mur ). A rapid induction in JA level following inoculation with B. cinerea was observed in Arabidopsis plants accumulating NO (Hebelstrup ). Interactions between NO and JA signaling have only been investigated during post-embryonic growth, with no information related to embryogenesis. Here we show that NO influences the number of somatic embryo produced by modulating the JA level and response, and that these effects can be integrated in the PGB2 regulation of somatic embryogenesis (Elhiti ).

Reports on exogenous JA applications during in vitro embryogenesis are scarce and contradictory. While promoting the formation of protocorm bodies, the equivalent of somatic embryos in orchids (Teixeira da Silva, 2012), applications of JA repress callus growth and somatic embryogenesis in Medicago sativa (Kepezynska and Zielinska, 2006). These discrepancies might be ascribed, at least in part, to differences in endogenous JA content, as also observed in our system. Applications of JA (50nM) enhanced embryo production in the WT line, characterized by a low JA signal in the embryogenic tissue, but not in the pgb2 line accumulating high levels of JA and producing a higher number of embryos (Figs 1C, 2). In this latter line, a depletion in JA level by Ph, an effective lipoxygenase inhibitor of JA synthesis (Farmer ; Bruinsma ), reduces embryo number, and this reduction is not due to the toxic effect of the inhibitor since embryo production is re-established by JA+Ph (Fig. 1C). Therefore, the enhanced embryogenic performance of the pgb2 line is most probably the result of a higher JA content within the embryogenic tissue.

Jasmonic acid mediates the NO and PGB2 regulation of somatic embryogenesis. First, the promotive effect of high NO environments (by SNP or suppression of Pgb2) on embryo formation is abolished by depleting JA with Ph. Consistent with this result, the reduction in embryo number under conditions of low NO (by cPTIO) is fully reversed by applications of JA (Fig. 2A). Secondly, manipulations of the NO content influence JA levels in the embryogenic tissue, with SNP increasing the intensity of the signal and cPTIO reducing it (Fig. 2B). Thirdly, NO induces the expression of two important JA biosynthetic genes, LOX2 and AOS, at both day 3 and day 7 on the induction medium (Fig. 3A), corresponding to the appearance of the embryogenic tissue (arrows in Fig. 1A). Among the 13 LOX genes found in Arabidopsis, LOX2 is the major contributor (~75%) to the total JA produced after wounding (Bell ; Schommer ). A similar result was also obtained for AOS, which catalyzes the dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Suppression of this gene almost completely abolishes the accumulation of JA following wounding (Park ; Von Malek ).

Unequivocal evidence for the involvement of JA as a downstream component of the NO mediation of somatic embryogenesis is apparent from the behavior of the JA-deficient aos1-1 line. Suppression of AOS reduces embryo production and this effect cannot be reversed by treatments altering NO levels, including SNP which under normal circumstances increases embryo production (Fig. 3B). The identical response observed in the pgb2/aos1-1 double mutant line, showing a reduction in embryogenesis independent of NO levels (Fig. 3C), confirms that JA is an integral mediator of NO generated by the suppression of PGB2.

The majority of JA responses observed during pollen development, wounding, and biotic stresses rely on a complicated interaction of downstream elements including JAZ1 and MYC2 (Kazan and Manners, 2013). In an uninduced state, the JA response is subdued by several repressors which are rapidly polyubiquinated and degraded via the 26S proteasome degradation pathway following JA treatments (Chini ). A rise in JA induces MYC2, a bHLH domain-containing transcription factor (Dombrecht ), which initiates a transcriptional cascade characterizing the ‘early JA response’. Within this cascade, genes participating in insect defense/wounding responses are up-regulated (Guerineau ), while others regulating auxin biosynthesis and responses to pathogens are repressed (Turner ; Wasternack and Hause, 2013) (diagram in Supplementary Fig. S7). The induction of MYC2 is transient, lasting only a few hours, as MYC2 also up-regulates its own repressor, JAZ1 (Chini ). Suppression of MYC2 by JAZ1 triggers the ‘late JA response’ characterized by the repression of the genes VEGETATIVE STORAGE PROTEINS 1 and 2 (VSP1 and VSP2), induction of the PLANT DEFENSIN 1 (PDF1) gene through the repression of the AP2/ERF transcription factor ORA59 and ETHYLENE RESPONSIVE FACTOR1 (ERF1), and the restoration of auxin synthesis through the de-repression of PLETHORA 1 and 2 (PLT1 and PLT2; Supplementary Fig. S8) (Kazan and Manners, 2013). Therefore, the temporal separation of ‘early’ and ‘late’ JA responses is controlled by the MYC2–JAZ1 feedback regulatory mechanisms (diagram in Supplementary Fig. S7).

Several pieces of evidence presented here suggests that an ‘early’ and a ‘late’ JA response occurs during somatic embryogenesis (as denoted by the biphasic profile of MYC2 expression, and the late increase in JAZ1 expression following JA treatments; Supplementary Fig. S4). Furthermore, the increased number of embryos produced by the extended applications of JA in the induction medium is possibly the consequence of the ‘late’ response characterized by the repression of MYC2 (Fig. 5; Supplementary Fig. S4), the increase in JAZ1 expression (Fig. 4; Supplementary Fig. S4), and the characteristic expression profile of genes involved in insect defense, pathogen response, and auxin synthesis (Supplementary Fig. S7). Within the ‘late’ response, suppression of MYC2 and up-regulation of JAZ1, possibly through a feedback mechanism (with MYC2 inducing JAZ1 and JAZ1 repressing MYC2) (Fig. 6), are essential for enhanced embryogenic output. Suppression of MYC2 directly via JA or indirectly via NO (by SNP treatments or suppression of PGB2) (Fig. 5A) increases the number of Arabidopsis somatic embryos (Fig. 5C). Consistent with this behavior, treatments reducing JA levels (by cPTIO or by suppression of AOS) up-regulate MYC2 (Fig. 5B) and compromise embryogenesis. The inefficacy of NO and JA manipulations to reduce embryo yield in a myc2-1 background confirms MYC2 as a downstream component of the NO and JA response (Fig. 5C).

Unlike MYC2, a direct or indirect rise in JA (by SNP or suppression of PGB2) induces JAZ1 especially within the cotyledons of the explants producing embryogenic cells (Fig. 4B) and favors the production of somatic embryos (Fig. 4D). The expression of JAZ1 is repressed in environments depleted in JA (by cPTIO or by suppression of AOS), a condition inhibiting embryogenesis (Fig. 4A, C). As also observed for MYC2, NO and JA treatments have no effects on the embryogenic performance of the jaz1-1 line (Fig. 4D), thus placing JAZ1 as downstream of NO and JA.

The beneficial effects of MYC2 suppression and JAZ1 induction on somatic embryogenesis are linked to a rise in the level of auxin, the inductive signal triggering the de-differentiation of the cotyledon cells and the production of embryogenic tissue (Raghavan, 2004). Two key transcription factors linked to auxin production, PLETHORA 1 and 2 (Pinon ), as well as several auxin biosynthetic genes including ASAI, converting chorismate to ribosyl anthranilate during the early steps of IAA synthesis, YUC4, converting indole-3-oyl pyruvate to (indol-3-oyl)acetate, and AMI1, producing IAA from indole-3-acetamide (Pollman ), are induced in NO- and JA-enriched environments and repressed by conditions depleting NO and JA (Fig. 7; Supplementary Figs S5, S6, S8). The observation that their expression is increased by JA (regardless of the levels of NO, suppression of MYC2, or induction of JAZ1) places these genes downstream of the NO response mediated by JA. While the involvement of MYC2 in the regulation of these genes during embryogenesis confirms previous studies (Elhiti ), that of JA and JAZ1 is novel and opens up new avenues for improving propagation methods in recalcitrant species. The embryogenic tissue-specific localization pattern of both ASA1 and YUC4 (Supplementary Figs S5, S6) suggests that regulation of these genes might be specific to the acquisition of embryogenic competence of the cotyledon cells, and might be related to the high levels of IAA observed in these areas (Fig. 8).

Collectively, these data suggest that mechanisms modulating late JA responses post-embryonically are integrated in the PGB2 and NO regulation of in vitro embryogenesis. In the proposed model (Fig. 9), a rise in NO due to suppression of PGB2 (or pharmacological treatments) induces the expression of the JA biosynthetic genes LOX2 and AOS and elevates JA along the cotyledons of the explants generating embryogenic tissue. JA evokes a response suppressing MYC2 and inducing JAZ1. This response, possibly reinforced by transcriptional feedback mechanisms between the two genes, up-regulates several auxin biosynthetic genes, leading to an increase in auxin levels. Auxin is required for the formation and proliferation of the embryogenic tissue, and ultimately for the successful production of the somatic embryos.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Characterization of the pgb2/aos1-1 double mutant lines.

Figure S2. Characterization of the 35S:PGB2/pgb2 line

Figure S3. Expression of jamsonic acid- and auxin-related genes in the 35S:PGB2/pgb2 line

Figure S4. Effects of JA applications on myc2-1 and jaz1-1.

Figure S5. Jasmonic acid (JA) and nitric oxide (NO) affect ASA1 expression and localization.

Figure S6. Jasmonic acid (JA) and nitric oxide (NO) affect YUC4 expression and localization.

Figure S7. Diagram showing the early and late JA responses.

Figure S8. Expression levels of PLETHORA 1 and 2 in day 7 explants.

Table S1. Primer list.