Recomendaciones para la implementación futura de la NC ISO 50001 (2011)

To identify the strongest gene responses affected by varying levels of N and rhizobium inoculation, the fold change of the DEGs were analysed after applying a cut-off +1.5 and -1.5 and p-value 0.05. All the DEGs were assigned to 12 groups of samples that represents of (i) deplete and replete N (ii) rhizobium presence or absence samples in A17 and sunn-1 (Table 3.1.3). Patterns of comparisons between treatments were generated separately for the defined groups to identify the strongest responsive genes as (i) Rhizobium effect, (ii) N effect and (iii) genotype effect. All the treatment sample groups and abbreviations used for classifying the groups (Table 3.1.3) for FC comparison are given in (Table 3.1.3).

For rhizobium effect (Table 3.1.3) in A17 deplete N (i.e. N deprived samples treated with 0.1 mM N) FC was analysed between mock treated AMLL and ARLL in A17, and SMLL and SRLL in mock treated sunn-1. Similarly, rhizobium effect at replete N (N deprived samples treated with 5 mM N) was calculated with FC between AMLH and ARLH in A17, and SMLH and SRLH in sunn-1. For the N effect (Table 3.1.3) in absence or presence of rhizobia, FC comparison between mock treated plants of A17 at deplete and replete N was calculated (AMLL and AMLH), rhizobia-treated plants A17 as (ARLL and ARLH). Similarly, in sunn-1 mutant FC comparison for N effect was calculated as (mock, Rhizobium absence- SMLL and SRLH) and (SRLL vs SRLH).

For the genotype effect (Table 3.1.3) FC patterns were calculated between AMLL and SMLL (sunn-1 mutant effect in depleted N, -Rhiz), ARLL and SRLL (sunn-1 mutant effect in depleted N, +Rhiz), AMLH and SMLH (sunn-1 mutant effect in replete N, -Rhiz), between ARLH and SRLH (sunn-1 mutant effect in replete N, +Rhiz) (data not shown).

Overall, rhizobium had stronger effect than N effect for both the genotype with greater number of differentially expressed genes of FC >1.5 or FC<-1.5 (Figure 3.1.3 A). Out of a total of 1099 strong-responding DEGs in A17, 96% (1066) were in rhizobia-inoculated plants. 502 genes in A17 showed differential expression when rhizobium was inoculated in an N deplete background (487 upregulated, 15 downregulated) and 558 genes in A17 were differentially expressed in an N replete background (552 genes upregulated, 6 genes downregulated) (Figure 3.1.3 B). The hypernodulant sunn-1 mutant also showed a similar response with a prominent rhizobium effect during N depletion and repletion (Figure 3.1.3 A, B). Around 95% of DEGs (2280 genes out of 2396 DEGs) displayed a strong effect of rhizobium. However, the combinatorial effect of N and rhizobium in both the genotype was always stronger during N repletion than in N depletion (Figure 3.1.3 B).

Table 3.1.3: Group of samples that were compared together to calculate fold changes (Also in figure 3.1.3-B representing groups from 1-8). FC of the expression values of the differentially expressed genes between different samples (as indicated in the table) was calculated to identify genes responding to the effects listed in the table.

Response FC Calculated between samples

A17 sunn-1

Replete N effect in -rhizobia Group 1 AMLL vs AMLH

Group 2 SMLL vs SMLH Replete N effect in +rhizobia Group 3

ARLL vs ARLH SRLL vs SRLH Group 4 Effect of rhizobia at deplete N Group 5

AMLL vs ARLL SMLL vs SRLL Group 6 Effect of rhizobia at replete N Group 7

AMLH vs ARLH

Group 8 SMLH vs SRLH SUNN effect at deplete N and -

rhizobia

Group 9 AMLL vs SMLL SUNN effect at deplete N and

+rhizobia ARLL vs SRLL Group 10

SUNN effect at replete N and -

rhizobia AMLH vs SMLH Group 11

SUNN effect at replete N and

+rhizobia ARLH vs SRLH Group 12

When the N effect is considered, A17 and sunn-1 showed a higher number of downregulated genes during N repletion than in N depletion in the absence of rhizobium (23 downregulated genes in A17, 91 downregulated genes in sunn-1) (Figure 3.1.3 B). N repletion in the absence of rhizobium had the highest effect in sunn-1 with differential expression of 97 genes (91 downregulated, 6 upregulated) (Figure 3.1.3 B).

Strong responsive genes in A17 Strong responsive genes in sunn-1 31 462 88 2 4 0 0 0 14 4 0 1 2 5 3

Replete N effect in −Rhizobia Replete N effect in +Rhizobia Rhizobia effect in deplete N Rhizobia effect in replete N

Replete N effect, - Rhizobia Replete N effect, +Rhizobia Rhizobia effect, in replete N Rhizobia effect, in deplete N 325 716 330 3 80 0 8 5 7 6 0 1 2 3 0

Replete N effect in −Rhizobia Replete N effect in +Rhizobia Rhizobia effect in deplete N Rhizobia effect in replete N

Replete N effect, - Rhizobia

Replete N effect, +Rhizobia Rhizobia effect,

in deplete N Rhizobia effect,in replete N

(A)

(B) Rhizobia effect, replete N, sunn

Rhizobia effect, deplete N, sunn Replete N effect, +Rhizobia, sunn Replete N effect, -Rhizobia, sunn Rhizobia effect, replete N, A17 Rhizobia effect, deplete N, A17 Replete N effect, +Rhizobia, A17

Replete N effect, -Rhizobia, A17 Upregulated genes

Downregulated genes 826 646 17 6 552 487 1 5 319 489 2 91 6 15 10 23

Fig 3.1.3: (A) Four set Venn diagram showing unique and shared genes that are

strongly responsive to N and rhizobia in A17 and sunn-1. Sample groups represent highly regulated genes with FC >1.5 and FC <-1.5 (p-value < 0.05) in A17 and sunn-1 for- replete N responsive genes in the absence of rhizobium; replete N responsive genes in the presence of rhizobium; rhizobia responsive genes in deplete N; rhizobia responsive genes in replete N. Also see table 3.1.3- 1 . (B) Total number of DEGs that show significant upregulation or downregulation in A17 or sunn-1. The strongest responsive genes in each of the groups were selected based on FC >1.5 and FC <-1.5 to identify rhizobia effects, N effects and genotype effects in A17 and sunn-1 from the sample group presented in table 3.1.3-1. FCs were calculated as (1) AMLH-AMLL; Group 1 (Replete N effect, - rhizobia in A17) (2) SMLH-SMLL; Group 2 (Replete N effect, -rhizobia in sunn) (3) ARLL-ARLH; Group 3 (Replete N effect, +rhizobia in A17) (4) SRLL-SRLH; Group 4 (Replete N effect, +rhizobia in sunn) (5) ARLL-AMLL; Group 5 (Rhizobia effect, deplete N in A17) (6) SRLL-SMLL; Group 6 (Rhizobia effect, deplete N in sunn) (7) ARLH-AMLH; Group 7 (Rhizobia effect, replete N in A17); (8) SRLH-SMLH; Group 8(Rhizobia effect, replete N in sunn). Genotype effect data for groups 9-12 not shown.