C-terminal region of Os GAD4 has the ability to bind to Ca 2+ /CaM
Eight amino acid sequences in the C-terminal regions of GADs from 3 plant species were compared (Fig. 1a). A Ca2+/CaMBD is found in almost all plant GADs in the C-terminal region, and it was first reported in Petunia hybrida GAD (PhGAD) to modulate the GABA synthesis in plants (Baum et al. 1993). On the other hand, the existence of the rice GAD2 isoform lacking a CaMBD also has been found (Fig. 1a) (Akama et al. 2001). As shown in Fig. 1a, very little similarity of the C-terminal extension in plant GADs is observed, but they have some common features, including several conserved clusters in each GAD. In particular, a highly conserved tryptophan (W) residue is found at the central position of the domain. Moreover, 2 to 4 lysine (K) and arginine (R) clusters can be seen at the N-terminus and C-terminus of GAD. The tryptophan (W) residue and lysine (K) clusters have crucial roles in binding CaM to the CaMBD (Arazi et al. 1995). According to a report by Yap et al. 2003, E476 and E480 work as pseudosubstrates of glutamic acid (Glu) in Petunia GAD and have autoinhibitory functions in the absence of Ca2+/CaM binding. Results such as these leave us in no doubt that the Glu residues are conserved in all the C-termini of rice isoforms, excluding OsGAD2, indicating a probable conserved function. Between two tea GADs (CsGAD), CsGAD2 does not have the ability to bind to a CaM but it is upregulated by mechanical stress, whereas CsGAD1 can bind to CaM (Mei et al. 2016). From the sequence alignment, CsGAD2 lacks one Glu pseudosubstrate residue and one Lys (K) residue at the C-terminus, whereas CsGAD1 has the conserved motifs. The in vitro Ca2+/CaM binding abilities of PhGAD, OsGAD1 and OsGAD3 have been shown (Baum et al. 1993; Akama et al. 2001; Akama et al. 2020), we anticipated that OsGAD4 potentially has the same ability, because it contains all the characters that are required for binding to Ca2+/CaM. It was speculated from the structural features of OsGAD4-CaMBD that OsGAD4 is a common plant GAD that demonstrates Ca2+/CaM-dependent activation (Fig. 1a). In a-helical wheel analysis, all of the hydrophobic residues are grouped on one side of PhGAD, whereas residues with positive charges are on the opposite side (Fig. 1b). For effective CaM binding to the CaMBD of PhGAD, the Trp (W) residue and Lys (K) cluster are essential, which contribute to hydrophobic and electrostatic interactions, respectively (Arazi et al. 1995). At these crucial positions, OsGAD4 was identical to PhGAD, whereas OsGAD2 had a unique structure. In the CsGADs, CsGAD1 retained the basic residues for CaM binding, but CsGAD2 is missing one Lys and one Glu, as observed in the sequence alignment. This α-helix analysis in Fig. 1b supports the assumption of Ca2+/CaM binding ability of OsGAD4.
From our study, it is evident that OsGAD4 has an authentic CaMBD, where the C-terminal region can bind to Ca2+/CaM (Supplementary Fig. S1). In vitro experiments demonstrated that like typical GADs in plants, OsGAD4 is capable of binding Ca2+/CaM, suggesting that OsGAD4 is indeed a Ca2+/CaM-dependent enzyme.
In vivo truncation of the C-terminal region of Os GAD4-CaMBD by genome editing
The structure of the OsGAD4 gene including exon/intron positions is presented in Fig. 2a, where the position of the CaMBD is presumed to be in the proximal region of the last exon. To remove the C-terminal extension of OsGAD4-CaMBD, guide RNAs (gRNAs) were designed, as shown in Fig. 2b. We predicted that the upstream and downstream cleavage of F1 and R1 will result in a 216 bp deletion (Fig. 2b). Subsequent DNA repair caused the deletion of almost the entire CaMBD and permitted the production of GAD4 protein without the C-terminal domain.
Agrobacterium -mediated plant transformation and screening after regeneration
Transformation of rice scutellum-derived calli was done using Agrobacterium. A binary vector harboring gRNAs and the Cas9 gene cassette was introduced into the Agrobacterium strain. In total, 27 independent transgenic lines (T0) were obtained after the regeneration process.
Ten lines with shorter bands at around 180 bp along with the wild-type band 400 bp were selected as candidate lines after genome editing (Supplementary Fig S2). These lines were grown for further confirmation. Then, seeds were harvested and grown to analyze targeted genome editing and GABA content determination.
PCR analysis of DNA extracted from the candidate transgenic lines showed three different patterns of amplified bands (Fig. 3). The first (A type) had a band at approximately 400 bp similar to wild-type Nipponbare (Ni; designated W type); second (B type) with two bands at 400 bp and at approximately 180 bp (designated bi-allelic type); and third pattern (C type) with only a band at approximately 180 bp. The latter was the expected amplicon size for the genome-edited lines, resulting from the deletion of 216 bp. Moreover, it was notable that C type bands (180 bp) were only derived from the next generation of one T0 line, whereas the remaining 9 lines produced only A and B type bands. Among the 3 different band patterns, A represents W type GAD4 mutants, B represents the heterozygous condition (bi-allelic) and C indicates the expected homozygous mutant i.e., genome-edited bands (Fig. 3). Twelve seedlings (T1) of same panicle derived from the T0 generation were analyzed by PCR. Seedling numbers 1, 2, 3, and 7 were found to be W type; number 5, 6, and 9 were heterozygous B type, and numbers 4, 8, 10, 11, and 12 had the expected band size for homozygous genome-edited lines, C type.
After a series of transformation events, we obtained 259 T1 transgenic plants in total. Out of these, 114 plants were W type (44%), 75 plants were B type (29%), and 70 plants were C type (27%) (Supplementary Table 1).
To confirm the genome editing, we have performed DNA sequencing in the T1 generation and analyzed the sequence in comparison with a Ni reference. Wild-type Ni indicates the reference nucleotide (Fig. 4a); sequence analysis resulted in different types of genome-edited patterns in numbered 1 to 6. Here, sequence number 1 corresponds to type C, and sequence number 2 corresponds to the slower migrating band in type B (Fig. 4b). The remaining sequences are all W type with various sequence patterns. We observed alteration in amino acid sequence of wild-type Ni and genome-edited lines in the target region of CRISPR/Cas9 (Fig. 4c). Nucleotide sequences 1 to 6 shown in Fig. 4b correspond to translated polypeptide chains GE 1 to GE 6 in Fig. 4c, respectively. The deletion of the CaMBD resulted in a peptide with 9 artificial amino acids after the authentic N-terminal VVAN. The other mutations included longer polypeptide chains.
In vitro enzyme activity from total protein extracted from WT and #14 − 1 seeds
To examine GAD enzymatic activity at physiological pH in the presence or absence of Ca2+/CaM, we isolated a crude protein extract from rice grains, which was utilized in the GAD enzymatic reaction. Crude protein of #14 − 1 exhibited higher GAD activity compared with its intact authentic extract, possibly because of higher activity of GAD4ΔC in #14 − 1 (Fig. 5). At physiological pH (pH 7), Ca2+/CaM induced 1.3-fold higher activity in wild-type Ni, whereas GAD activity was higher in #14 − 1 with or without Ca2+/CaM. GAD activity increased 2.3- and 2.2-fold in #14 − 1 at pH 7 in the absence or presence of Ca2+/CaM in comparison to wild-type control without Ca2+/CaM. This implies that the C-terminal domain functions as a potential autoinhibitory domain in OsGAD4; as a result, when this domain is truncated, the enzyme acts constitutively and exhibits increased activity.
Measurement of free amino acids
The concentration of free amino acids was measured using gas chromatography-mass spectrometry (GC/MS) (Table 1). The quantities of asparagine (Asn) and Trp were significantly lower in the genome-edited line compared with the wild-type, whereas most other free amino acids levels were increased. Among the proteinaceous amino acids, the accumulation of valine (Val), isoleucine (Ile), leucine (Leu), Glu, and phenylalanine (Phe) was significantly higher compared with wild-type Ni. The highest GABA content was found in the line #14 − 1, which was almost 9-times higher compared with wild-type Ni. Line #14 − 1 samples corresponded to DNA sequence pattern type C (truncation of CaMBD). In the next generation (T2), we focused on the highest GABA yielding line derived from #14 − 1, and more seed was produced for further study.
Table 1
The concentration of free amino acids in rice grains (T2) (nmol/g grain), was determined by GC/MS.
Amino acid | Ni | #14 − 1 |
Ala | 56.1 ± 4.1 | 113.2 ± 7.1** (2.0) |
Gly | 15.9 ± 8.3 | 24.8 ± 1.6 (1.6) |
Val | 7.3 ± 0.8 | 39.1 ± 2.1** (5.4) |
Leu | 2.4 ± 0.6 | 12.9 ± 0.2** (5.3) |
Ile | 2.3 ± 0.1 | 12.2 ± 0.4** (5.4) |
Ser | 15.9 ± 1.5 | 31.1 ± 1.6** (2.0) |
Pro | 13.7 ± 2.8 | 8.7 ± 3.8 (0.6) |
Asn | 134.2 ± 15.0 | 44.8 ± 12.1** (0.3) |
Asp | 92.1 ± 26.0 | 271.4 ± 21.6** (2.9) |
Met | 42.4 ± 7.1 | 110.3 ± 9.7** (2.6) |
Glu | 149.0 ± 7.8 | 804.6 ± 32.2** (5.4) |
Phe | 2.1 ± 0.1 | 9.6 ± 1.0** (4.5) |
Gln | 22.2 ± 1.6 | 48.0 ± 4.4** (2.2) |
His | 17.7 ± 2.4 | 76.8 ± 9.1** (4.3) |
Tyr | 7.6 ± 5.0 | 2.3 ± 1.2 (0.3) |
Trp | 8.6 ± 1.0 | 1.7 ± 0.4** (0.2) |
GABA | 14.7 ± 1.2 | 129.8 ± 20.9** (8.8) |
Value: average ± standard deviation |
*P < 0.05, **P < 0.01 versus Ni control; Data in parentheses are fold change in comparison to wild-type Ni. |
GABA content in vegetative tissues was measured using a GABase assay. GABA concentration in line #14 − 1 was compared with wild-type Ni (Supplementary Fig. S3). Stem and root tissues had higher GABA contents than wild-type Ni. Among the vegetative tissues of line #14 − 1, root tissues accumulated the highest GABA levels.
Abiotic stress induced GABA accumulation in vegetative tissues
Earlier studies demonstrated that abiotic stresses increase endogenous GABA concentrations in plant tissues, although the rate of its accumulation varied widely (Li et al. 2021). We observed the responses to stresses in rice seedling at an early vegetative stage. Firstly, salt stress treatment of 2-week-old rice seedlings was performed in vitro using 150 mM NaCl for various time periods. Secondly, flooding stress treatment of rice seedling was performed by completely submerging the seedlings in liquid MS media, and finally seedlings at the same age were allowed to grow without any media for drought stress treatment.
Salt stress induced accumulation of GABA in shoot tissues of line #14 − 1 after 1 h of treatment, which was higher than wild-type Ni (Fig. 6a). GABA levels were reduced after 3 h of treatment, and levels climbed again at the highest point after 6 h of stress treatment, reaching 2.6-fold higher compared with wild-type Ni. In root tissues, GABA induction demonstrated a gradual increase with the duration of stress and rose to maximum at 6 h, producing an extreme yield of almost 4.3-fold higher GABA levels compared with wild-type Ni.
With flooding treatment, GABA accumulation started to rise drastically in root tissues of line #14 − 1 and it reached at peak of approximately 3.3-fold higher GABA accumulation in compared with wild-type Ni at 1 h (Fig. 6b). After this, GABA levels declined at 3 h and 6 h of flooding stress. In shoot tissues, there was similar trend of GABA production, with flooding stress producing significantly higher GABA levels after 1 h.
Furthermore, drought stress was imposed in young rice seedlings to evaluate the response in GABA accumulation. Shoot tissues exhibited elevated GABA concentration in response to drought stress starting from 6 h, with a slight reduction at 12 h, and again showing greater accumulation at 24 h (Fig. 6c). At this time point, line #14 − 1 produced approximately 2.2-fold higher GABA compared with wild-type Ni. Additionally, in root tissues, drought stress induced a gradual rise in GABA content with time, reaching its maximum level at 24 h.
Taken together, it can be seen that GABA induction in vegetative tissues of young rice seedling showed diverse responses to different abiotic stresses, and GABA accumulation was augmented in vegetative tissues to respond to abiotic challenges.
mRNA expression levels are upregulated in vegetative tissues upon exposure to abiotic stresses
To explore changes in the expression levels of OsGAD4, reverse transcription quantitative polymerase chain reaction (RT-qPCR) of vegetative tissues was done at different time points while exposed to the same abiotic stresses as for GABA content measurement. Both wild-type Ni and line #14 − 1 were analyzed but having the same promoter region they exhibited almost identical expression levels. For a better understanding, we show the data of line #14 − 1 only. In the case of salt stress, mRNA expression levels were induced significantly in root tissues at 1 h, then a slight drop at 3 h (Fig. 7a). Strong expression at 6 h reaching its peak at 3.1-fold compared with the untreated control. Similarly, shoot tissues showed consistent expression levels as root tissues, with comparatively lower expression levels at the same time point in response to salt stress.
During flooding stress, mRNA expression levels were upregulated rapidly at 1 h, with an approximately 3.1-fold increase in root tissue compared with the untreated control (Fig. 7b). Surprisingly, in the same stress condition, shoot tissues did not show much upregulation, though levels were higher compared with the control. After this time point, the expression in root tissues declined slightly up to 3 h but then increase at 6 h. Again, a similar tendency was observed in shoot tissues, with a trend of declining expression at 3 h but then significantly increased expression at 6 h.
With respect to drought stress, both shoot and root tissues exhibited steady upregulation of expression at the onset of stress treatment at 1 h (Fig. 7c). At the 3 h time point, expression levels had a trend towards a decrease in shoot tissues, almost similar to untreated samples. However, root tissue showed increased expression at this point. Eventually, with an increase in duration of stress the expression level increased moderately and reached a maximum at 24 h. In shoot and root tissues, the increase was 7.5-fold and 6.3-fold, respectively, compared with the untreated control. Our mRNA expression data is mostly in compliance with the gene expression profile or transcriptional activity of the TENOR database (Kawahara et al. 2016).
Abiotic stress tolerance was significantly improved in genome-edited rice plants
The tolerance level of OsGAD4 genome-edited plants to salt, flooding, and drought stresses were examined compared with that of wild-type plants. Two-weeks old seedlings were treated with multiple stresses as described in the material and method section. After stress treatment, the seedlings were washed and rehydrated for 3 h before transferring to small pots containing soil (Fig. 8). Salt stress tolerance in line #14 − 1 was demonstrated in our experiment using 250 mM NaCl solution. The survival rate of line #14 − 1 was 41.6% against salt stress, whereas 16.6% of wild-type plants survived the stress (Table 2). For flooding stress, 24.8% of wild-type plants survived, and line #14 − 1 showed a survival rate of 58.4%. With drought stress, 33.3% of line #14 − 1 seedlings endured the stress, whereas the survival rate of wild-type was only 12.5% (Table 2). Our results showed that the genome-edited mutant of OsGAD4 significantly improved abiotic stress tolerance at the early vegetative stage.
Table 2
Quantitative data on survival rate and biomass reduction after the seedlings survived abiotic stresses.
Type of stress | Survival rate (%) | Biomass loss % (FW) | Biomass loss % (DW) |
Ni | #14 − 1 | Ni | #14 − 1 | Ni | #14 − 1 |
Salinity | 16.6 ± 8.8 | 41.6 ± 11.8** | 31.6 ± 3.7 | 11.0 ± 4.2** | 29.5 ± 16.3 | 13.6 ± 8.7* |
Flooding | 24.8 ± 5.9 | 58.4 ± 21.5* | 27.1 ± 8.4 | 9.9 ± 1.3** | 24.9 ± 5.5 | 10.9 ± 7.3** |
Drought | 12.5 ± 5.9 | 33.3 ± 11.8** | 33.1 ± 3.0 | 14.7 ± 3.7** | 28.7 ± 3.3 | 9.9 ± 2.1** |
DW = dry weight, FW = fresh weight. The values are calculated from averages of the surviving plants in each stress with wild-type and line #14 − 1. The average number of surviving plants were converted to percentage to show the survival rate. These data represent the combined result of two independent experiments each with three replications in each stress condition. In the case of biomass, the control or untreated fresh and dry weight was assumed as 100%. Biomass loss was calculated by deducting the biomass of stress surviving plants from that of the untreated one. Asterisks indicate significant differences compared to wild-type (*P < 0.05, **P < 0.01). |
Biomass comparison was done by measuring the fresh weight and dry weight of the surviving plants. Fresh weight was measured after removal from soil and washing to remove dirt. For measurement of dry weight, the cleaned plant sample was dried overnight in an oven at 45°C. Results revealed that biomass reduction as fresh weight in #14 − 1 plants in the three stress conditions was significantly lower than that in the wild-type plants (Table 2). Similarly, dry weight loss was more in wild-type plants compared with #14 − 1 plants in response to the abiotic stresses, suggesting a protective mechanism enabled #14 − 1 plants to tolerate the adverse conditions.
Our current results indicated that elevated GABA levels are possibly associated with abiotic stress tolerance in plants by playing a protective role. The underlying mechanism needs to be studied further.