## 2 Results
### Proteome Profiling of Macro-Mutant Lines
#### 2.2.2 Identification and Expression Patterns of Differentially Expressed Proteins of Mutant lines  
The differentially expressed protein spots were identified, as shown in Table 4. The up-regulation and down-regulation of these proteins (compared with the control) are depicted in Table 5. Spot #1011 was identified as a large subunit of ribulose bis-phosphate carboxylase/oxygenase. The nominal mass and isoelectric point of this spot were 50,841 Da and 6.22, respectively. This protein is chloroplastic and is involved in the photosynthetic process. The expression of this protein was upregulated in mutant HY1, MHY2, and LY1, when compared to the control. In LY3, VLY2, and VLY3, the expression \begin{table}
\begin{tabular}{c c c} \hline \hline
**S.N.** & **Control/Mutants \({}^{*}\)** & **Number of Protein Spots** \\ \hline
1. & Control & 241 \\
2. & HY1 & 258 \\
3. & HY3 & 238 \\
4. & MHY2 & 222 \\
5. & MHY4 & 260 \\
6. & NY3 & 249 \\
7. & LY1 & 219 \\
8. & LY3 & 255 \\
9. & VLY2 & 251 \\
10. & VLY3 & 220 \\ \hline \hline \end{tabular} \({}^{*}\) Abbreviations of mutant lines are as per Supplementary Table S2.  
\end{table} 
Table 2: Total number of the leaf protein spots of _Nigella sativa_ L.  
Figure 1: Representative 2-DE profiles of the leaf proteins of _Nigella sativa_ L.  
of this protein in the leaves was downregulated compared to the control plants. Expression of this protein in HY3 and NY3 was equal to the control. Spot #3401 was identified as ribulose bisphosphate carboxylase/oxygenase activase. The molecular weight (MW) and isoelectric point (pl) of this spot were found to be 48,079 Da and 6.20, respectively. This is a chloroplastic protein, found in the stroma. The rubisco activase is involved in the process of photosynthesis, through the activation of rubisco. The pattern of the expression of this protein in the leaves of different mutants of _N. sativa_ was similar to that of spot #1011, except for NY3, where the expression of this protein was downregulated. Spot #4201 was the ATP synthase gamma chain. The MW and pI of this spot were found to be 41,706 Da and 8.1, respectively. It is also an ATP synthase subunit, involved in the synthesis of ATP. Compared to the control, the expression of this protein is upregulated in the leaves of the HY1, HY3, MHY4, NY3, LY3, and VLY2 mutants. In mutants MHY2, LY1, and VLY3, the expression of this protein is equal to that of the control plant (Tables 4 and 5). Spot #1201 has been identified as oxygen evolving enhancer protein 1. The MW and pI of this spot were found to be 35,377 Da and 5.5, respectively. This protein is also a part of the photosynthetic pathway. It is located in the thylakoid membrane of the chloroplast. This protein stabilizes the manganese cluster, which is the primary site of water splitting. The expression of this protein in the leaves of the HY1, MHY2, and LY1 mutant lines was higher than that of the control plants, but for LY3, VLY2, and VLY3, its expression was downregulated. There was no difference in the expression of this protein in HY3, MHY4, and NY3, when compared to the control. Spot #4301 has been identified as the PSII stability/assembly factor, HCF 136. The molecular weight and pI of this protein spot were found to be 45,498 Da and 9.0, respectively. This is also a chloroplastic protein that is involved in the photosynthetic process of plants. This is an essential protein for PS II biogenesis and is required for the assembly of an early intermediate in PS II. The expression of this protein was higher in the leaves of the HY1, MHY2, and LY3 mutants than in the leaves of the control plants. In all other mutants (HY3, MHY2, NY3, LY1, VLY2, and VLY3), the expression of this protein was equal to that of the control plants. Spot #2101 has been identified as imidazole glycerol-phosphate dehydratase 1. The MW and pI of this spot were found to be 29,264 Da and 7.2, respectively. The cellular location of this protein is in the chloroplast. It is involved in the biosynthesis of amino acids.