Integración del sistema tributario

Grain nitrogen concentrations differed significantly between the lines (P<0.01). R8 had a significantly higher nitrogen concentration than C, while the other lines had intermediate grain nitrogen concentrations (Figure 5.24.a). Total grain nitrogen content did not differ significantly between the lines. R5 had the highest grain N content (Figure 5.24.b), because it had the highest grain yield (Figure 5.23.a). Despite the low grain yield of R8 (Figure 5.23.a), its high grain N concentration resulted in the second highest N content (Figure 5.24). Over the whole experiment, grain N concentration correlated significantly with grain yield (P<0.001). However, at similar grain yields as control plants, the transgenic plants reached higher grain N concentrations (Figure 5.25; compare dashed and solid lines). The differences were greater at lower grain yields.

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Figure 5.22: Aboveground biomass per plant at anthesis and maturity of a selection of NAC-overexpressing lines of wheat. Data are means + SE of four replicate plants at anthesis (GS61) and nine replicate plants at maturity (GS92).

Figure 5.23: Grain yield per plant (a), straw and chaff yield per plant (b) and harvest index (c) of a selection of NAC-overexpressing lines of harvest-ready wheat. Data are means + SE of nine replicate plants.

0,0 10,0 20,0 30,0 40,0 50,0 60,0 Anthesis Maturity D ry w ei gh t (g) pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control 0,20 0,30 0,40 0,50 0,60

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control

H arv e st In d ex

c

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control

D ry w e igh t (g)

a

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control

D ry w e igh t (g)

b

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Figure 5.24: Grain nitrogen concentration (a) and total grain nitrogen content (b) of a selection of NAC-overexpressing lines of harvest-ready wheat. Data are means + SE of nine replicate plants. Letters indicate significant differences at the 0.05 level as determined by using the LSD.

Figure 5.25: Relationship between grain yield per plant (GY) and grain nitrogen concentration (GNC) of a control line (solid line) and of a selection of transgenic NAC-overexpressing lines of wheat (dashed line). Each data point represents one plant. Control: y = -0.0467x + 3.0323; R2 = 0.4389; P < 0.05. Transgenic: y = -0.0802x + 3.9532; R2 = 0.6662; P < 0.001. 0,0 1,0 2,0 3,0 4,0 5,0 0 10 20 30 40 G ra in N c o n ce n tra ti o n (% )

Grain yield per plant (g)

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control Control Transgenic ab ab ab a b 0,0 1,0 2,0 3,0 4,0

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control

N c o n cn tra ti o n (% )

a

pActin-NAC 1 pRTBV-NAC 4 pRTBV-NAC 5 pRTBV-NAC 8 Control

N c o n te n t (m g)

b

173 5.6 DISCUSSION AND CONCLUSIONS

The expression of the endogenous wheat NAC gene decreased with senescence (Chapter 3 and Figure 5.19.b), indicating the gene is a potential negative regulator of leaf senescence. NAC genes that are negative regulators of senescence seem to be quite rare: so far only the JUB1 gene has been unequivocally identified as a regulator of senescence (Wu et al., 2012). Apart from this example, a wheat gene showing strong homology with rice NAC2

was identified in a transcriptome study comparing GPC-RNAi with wild-type wheat during senescence (Cantu et al., 2011), but its function has not been determined.

NAC overexpression under the RTBV promoter resulted in delayed leaf senescence, whilst overexpression under the actin promoter did not alter leaf senescence in most cases (Figures 5.10, 5.11, 5.12 and 5.15). In the most detailed experiment, line R8 was the only line that maintained a higher chlorophyll content and / or photosystem II capacity than wild-type and control in all three top leaves (Figures 5.10, 5.11 and 5.12). Line R5 had especially green second leaves, while R5, R2, R3 R6 and R7 all had significantly higher chlorophyll content in the third leaf at some point during senescence. In another experiment, the top three leaves of R5 clearly stayed the greenest, while R4 and A1 also maintained greener than C (Figure 5.15). R8 performed similar to wild-type in the second experiment (Figure 5.15), while in the initial detailed experiment it was the line that clearly stayed the greenest for longest (Figures 5.10, 5.11 and 5.12).

The differences between the two experiments can partly be explained by environmental conditions, even in the relative constant conditions of the glasshouse. In the second experiment the plants experienced an unexpected hot and sunny week in March about one week after anthesis, inducing rapid senescence. In the first experiment senescence progressed more gradually,

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less influenced by fluctuations in temperature. The senescence data from the first experiment are therefore probably more reliable.

Grain yields and aboveground biomass were not significantly different between NAC-overexpressing and control lines (Figures 5.13, 5.22 and 5.23.a), although the most stable stay-green line R5 had the highest grain yield and seemed to have a relatively high harvest index (Figure 5.23.c). Surprisingly, the control line had the biggest post-anthesis biomass gain, suggesting that the stay-green lines did not have a higher post-anthesis photosynthetic production. These results confirm the hypothesis that grain yield is generally not source-limited, as has been found before for wheat, including stay-green varieties (Borrás et al., 2004; Gong et al., 2005).

Grain nitrogen concentration and content were higher for the four examined NAC-overexpressing lines than for the control plants (Figure 5.24). Transgenic plants reached higher grain N concentrations at similar grain yields, especially when grain yield was relatively low (Figure 5.25). Line R8 had the highest grain N concentration but the lowest grain yield, so its high N concentration was probably caused by less carbon dilution, although this cannot be known for certain without information about the grain number. Line R5 had the highest grain N content, which was due to its relatively high grain yield. Since control plants had the highest straw biomass (Figure 5.23.b), it can be speculated they invested relatively more nitrogen in the vegetative parts and less into the grain. However, nitrogen concentration and content data for the whole plant are required to be able to discriminate between a lower nitrogen harvest index and a lower N uptake.

Line R8 was dissimilar from the other lines in development and morphology: short in stature, initially few but late developing tillers, and fewer and wider leaves (Figures 5.5, 5.6, 5.7, 5.8, 5.9 and 5.13). In the first experiment R8 coupled an early developing main shoot with late maturation (Figure 5.5), and therefore had greater post-anthesis longevity. In the second experiment it

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reacted very strongly to a short peak in temperature, inducing fast senescence, suggesting it did not well tolerate stress conditions. Expression levels of the transgene did not seem much different from that of the other transgenic lines. It could be that the transgene was inserted into a gene or an active genomic region, affecting function or expression of other genes.

For the expression results it is important to remember that no gene expression data are available for the last time point for three of the lines. Expression in later stages of senescence is therefore difficult to compare. Line R4 had the highest NAC expression but not the strongest stay-green phenotype. This indicates that the level of overexpression was not directly correlated with the stay-green phenotype, but that expression probably had to be above a certain threshold. Transgenic NAC expression increased across the senescence time course, suggesting that the RTBV and actin promoters were more active in later stages of leaf development, or alternatively, that the transgenic RNA was not degraded very efficiently.

The observation that NAC overexpression under the actin promoter did not usually result in a phenotype is remarkable. The actin promoter is constitutively expressed (Zhang et al., 1991) and therefore should be expressed in leaves too, so it is not obvious why there was no effect. The RTBV promoter is supposed to be more strongly expressed in wheat leaves than the actin promoter (Rothamsted Cereal Transformation Group, unpublished), although this has not been confirmed throughout leaf development. Alternatively, the timing of expression in the leaves could be different. It is also possible that expression in other plant organs under the actin promoter interferes with the effect of the expression in leaf tissues.

Apart from the changes in leaf senescence, the overexpression lines did not show any other consistent change in phenotype. Therefore it is likely the function of the NAC gene is primarily in the regulation of leaf senescence. Expression analysis has shown the closest rice gene, Os08g10080, is

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differentially expressed in response to infection with the rice tungro spherical virus (Nuruzzaman et al., 2010) and mild drought stress (Nuruzzaman et al., 2012). This could indicate a function in stress responses, although the opposite expression pattern for different stresses contradicts this. Since orthologous NAC transcription factors in wheat and rice have been shown to have diverse functions (Distelfeld et al., 2012), the rice expression does not necessarily have relevance for the wheat gene. However, since many genes involved in senescence are also involved in stress responses, the response of the wheat NAC gene to different stressors would be an obvious topic for further investigation.

The greenness of leaves is a measure of leaf nitrogen content (Richardson et al., 2002). In agreement with this, the stay-green trait was found to be positively correlated to nitrogen use efficiency (Gaju et al., 2011). Therefore the response of the NAC-overexpressing lines to differences in nitrogen nutrition would be of interest.

It has been hypothesised that maintenance of N uptake during grain-filling is determined by the carbon supply to the roots and that this would result in a stay-green phenotype by less N remobilisation from the leaves (Rajcan and Tollenaar, 1999b). Furthermore, higher than expected grain N concentrations as found for the NAC-overexpressing lines (Figure 5.25) have been shown to be mainly determined by N uptake (Bogard et al., 2010). Therefore it would be interesting to know whether there are differences in N uptake between the lines, both pre- and post-anthesis, and whether such differences could be explained by differences in root biomass or morphology.

In summary, a NAC1-type NAC transcription factor with decreasing expression in leaf senescence of wheat has been identified. Overexpression of this NAC gene in wheat resulted in delayed leaf senescence and increased grain nitrogen concentrations, but had no significant effects on shoot biomass or grain yield.

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