Marginal impact of loss of major PIPs in leaves in response to water stresses at

During drought stress and heat stress, two of the most important water stresses, plant water relations are regulated through changes in abundance of aquaporins (Maurel et al., 2008).

The expression of PIPs is generally downregulated in leaves under drought stress except for

PIP1;4 and PIP2;5 (Figure 19), which is in agreement with previous studies (Alexandersson et

97 al., 2005; Alexandersson et al., 2010). The downregulation of PIPs in leaves, especially in bundle sheath cells, would lead to stomata closure to avoid excess water loss when the root water supply is restricted under drought stress in accordance to the consideration of Shatil-Cohen et al. (2011). However, Martre et al. (2002) and our results showed that the repression of expression of PIPs is not sufficient to reduce the transpiration rate and stomata aperture under well-watered condition or drought stress with low transpiration demand as compared to the wild type (Martre et al., 2002). This suggests that the repression of PIPs is a strategy of plants in response to drought stress, but not related to transpiration regulation.

Furthermore, our results showed that the drought-responsive genes in wild type as compared to control condition were generally less deregulated in pip2;1 pip2;2 (Table 9), suggesting loss of PIP2;1 and PIP2;2 alleviated the drought responses at transcriptional level.

Rae et al. (2011) found that the transcription factor RAP2.4B positively regulated the expression of PIPs. RAP2.4B was induced by heat stress, which indicated that PIPs may be upregulated in response to heat (Rae et al., 2011). In agreement with this study, the expression of PIPs were generally upregulated under regular heat stress except for PIP1;4, PIP2;5 and PIP2;8. In addition, PIPs were also generally induced to a similar expression level at H HrH, when the additional water deficit in air was eliminated and the transpiration was restricted (Figure 19). These results suggest that the heat effect predominantly contributes to the upregulation of PIPs and the upregulation of PIPs is irrespective to the transpiration under heat stress. To further explore the functions of upregulated major PIPs under heat stress, transcriptional changes in response to heat stress in pip2;1 pip2;2 were examined.

PIP2;1 and PIP2;2 are highly expressed in leaves and loss of these two major PIPs were

expected to aggravate the heat stress responses. Surprisingly, heat-responsive genes of the

wild type were changed to a similar extent in pip2;1 pip2;2 under either H LrH or H HrH as

compared to control condition (Table 10 and Table 11). Thus, the role of upregulation of

PIP2;1 and PIP2;2 in response to heat stress cannot be deduced from this finding. One

possibility is that the excited activities of the remained PIPs (e.g. PIP1;2, PIP2;6 and PIP2;7),

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which are expressed in veins, may partially complement the loss-of-function of PIP2;1 and PIP2;2. On the other hand, loss of PIP2;1 and PIP2;2 may only delay the optimal adjustment of leaf water relations, but not determine the final leaf water status. To support this hypothesis, several studies have shown that the regulation of PIPs expression responded quickly to changing environmental conditions (Siefritz et al., 2004; Levin et al., 2007; Caldeira et al., 2014b; Caldeira et al., 2014a). Rae et al. (2011) also showed that transcription factor RAP2.4B, which is targeted to PIP promoters, was highly induced after heating for 2 h, but started to decrease after heating for 6 h. So the dynamic changes of the water relations after heating could be more interesting to evaluate the PIPs functions. Therefore, regulation of PIPs along with the time after heating should be examined. Then the dynamic changes of leaf water potential and hydraulic conductivity should be measured in pip mutants and wild type to assess the functions of PIPs in regulation of water relations under heat stress.

Furthermore, the dynamic changes at transcriptional and metabolic levels along with the time after heating could be interesting to evaluate other impacted biological processes due to loss of PIPs in response to heat stresses.

Drought stress and heat stress have opposite effects on the regulation of expression of PIPs.

Therefore, it was interesting to study the regulation of PIPs under combined drought and heat stresses. Previous studies have shown that drought effects predominantly contributed to the response to a combined drought and regular heat stress scenario (Rizhsky et al., 2004).

In agreement with these studies, differential regulation of PIPs under DH LrH was similar to

that under drought stress (Figure 19). However, high relative air humidity enhanced the heat

stress responses and in turn aggravated the heat effect, but alleviated the drought effect

(see 3.1). Therefore, the differential regulation of PIPs under DH HrH generally tended to be

shifted from drought-related, suppressive effects to heat-related inductive responses (Figure

19). Among these PIPs, PIP2;1 and PIP2;2 were downregulated under DH LrH, similar to the

situation under drought stress. Therefore, loss of PIP2;1 and PIP2;2 under DH LrH was

expected to have impacts similar to those under drought stress. In agreement with our

99 expectation, the DH LrH-responsive genes were generally less deregulated in pip2;1 pip2;2 than in the wild type, which was also found in the pip double mutant under drought stress (Table 9 and Table 13). In addition, PIP2;1 and PIP2;2 were not changed under DH HrH (Figure 19) and the DH HrH-responsive genes of the wild type showed similar changes in pip2;1 pip2;2 (Table 12). Taken together, the regulation of PIPs under combined drought and heat stresses is determined by the predominant effect to allow optimal adjustment of the leaf water status under combined, yet antagonizing effects (Guyot et al., 2012).

On the other hand, marginal changes were detected at the transcriptional level in pip2;1 pip2;2 as compared to the wild type under various water stresses (Figure 30 and Table 14-18). Among these genes, QQS was upregulated in pip2;1 pip2;2 under all water stresses, suggesting that soluble sugars might be overaccumulated in the cytosol to regulate the osmotic potential (Li et al., 2009). In addition, cell wall modification was regulated by invoking different genes under various water stresses. The gene encoding FUCOSYLTRANSFERASE 4 (FUT4) functions in fucosylation of arabinogalactan proteins in leaves (Tryfona et al., 2014) and was downregulated under drought stress, suggesting a reduced mechanical strength of the cell wall (Reiter et al., 1993). The cell wall-associated genes CELLULOSE SYNTHASE-LIKE A1 (CSLA01), which is involved in biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2007) and LEUCINE-RICH REPEAT/EXTENSIN 1 (LRX1), which functions in cell expansion (Baumberger et al., 2003) were upregulated under DH LrH. These results suggest that the reduction of osmotic potential by overaccumulation of soluble sugars may enhance the driving force of water influx into cells and in turn support normal cell growth assisted with enhanced cell extensibility by modification of cell wall components in response to water stresses when loss of PIP functions.

In conclusion, our results revealed that loss of PIP2;1 and PIP2;2 slightly reduce the

transpiration water loss in leaves under well-watered condition and this reduction is

dependent on root water supply as well as leaf water demand. These findings were

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consistent with previous studies which showed that low relative air humidity increased the leaf hydraulic conductivity (Levin et al., 2007) and that drought stress mimicked by xylem-fed ABA reduced the leaf hydraulic conductivity (Shatil-Cohen et al., 2011) mediated by regulation of aquaporins. In addition, loss of PIP2;1 and PIP2;2 had no impact on leaf growth.

Kaldenhoff et al. (1998) had found that improved root growth compensated the reduced root hydraulic conductance to support the normal growth in Arabidopsis PIP1-antisense plants. In contrast, root growth was not impacted in pip2;1 pip2;2, but the cellular osmotic potential and cell wall plasticity could be modified to support the normal growth. On the other hand, PIPs were deregulated under various water stresses. Our results showed that the expression of PIPs were downregulated under drought stress as well as DH LrH when drought stress is the predominant effect, and loss of PIP2;1 and PIP2;2 alleviated the stress responses based on the lowered transcriptional deregulation in comparison to wild type. In contrast, the expression of PIPs was upregulated under heat stress, irrespective of differences in relative air humidity. But the regulation of PIP expression was relatively shifted towards a heat effect along with the aggravated heat stress under combined drought and heat stress with high relative air humidity. Surprisingly, loss of PIP2;1 and PIP2;2 had no impact on the response to heat stress and combined drought and heat stress with high relative air humidity. Furthermore, loss of PIP2;1 and PIP2;2 may result in osmotic potential regulation and cell wall modification to regulate the water status under water stresses, and these changes may compensate the loss of the functions of PIP2;1 and PIP2;2. However, this compensation was still weak, especially under heat stress responses when the two major PIPs were upregulated. We speculate that the upregulation of PIPs may be a fast response to heat stress and play a critical role in speed up the water homeostasis after heating.

Therefore, the dynamic changes in response to heat should be a focus of future research.

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4 MATERIALS AND METHODS