Régimen general de las Entidades colaboradoras

Besides the accumulation of storage proteins and lipids during seed maturation, stress-related proteins are also accumulated. LEA proteins and HSPs play essential protective functions during acquisition of dessication tolerance, at late maturation stage, preventing the macromolecules damage and promoting the maintenance of cellular stability. They are immobilised during early stages of seed germination, where some of them play protective roles and others are integrated in biosynthetic pathways. (Hong-Boa et al. 2005; Manfre et al. 2006; Kotak et al. 2007; Hundertmark and Hincha 2008; Manfre et al. 2009). As HRR is mainly expressed in later stage of seed maturation (stage 6, Figure 3.26A), it would be interesting and important to know if HRR is involved in regulation of transcript levels of some key stress-related proteins. For this, Em1 and Em6 (LEA proteins),

HSP101 and HSFA9 gene expression levels were analysed during seed development (Figure

3.31A). Their corresponding genes display considerable expression levels during later stages of maturation (Kotak et al. 2007; Bentsink and Koornneef 2008). When the environmental conditions (light, nutrients and temperatures) are ideal, seed will enter in the germination process and the accumulated stress-related proteins will be recruited, allowing the osmotic adaptation of germinating seed. For this, the same transcript levels were analysed during seed germination (Figure 3.31B).

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Figure 3.31 Expression analysis of LEA protein genes (Em1, Em6), HSP gene (HSP101) and seed-specific HSF

gene (HSFA9) during seed development and germination. Transcript levels were evaluated by semi-quantitative RT- PCR from mRNA extracted (A) from flower/silique tissues, according to defined seed development and maturation stages (Figure 3.25) or (B) from germinated seeds with one or two days upon sowing in MS-agar medium. For comparison experiments were performed in wild-type Ler and hrr mutant. As internal control, the transcript levels of Actin2 (Act2) were analysed. Numbers on the right correspond to the expected sizes of PCR products. The pair of primers and PCR conditions are described in Annexes III and IV, respectively.

Em proteins belong to group 1 of Late Embriogenesis Abundant (LEA) proteins, being expressed in later stages of embryo maturation (acquisition of dessication tolerance) and during water deficit in vegetative organs, suggesting a protective role during water limitation (Hundertmark and Hincha 2008). HSFA9 was described as a specialised HSF for embryogenesis and seed maturation, controlled by hormonal networks (ABA and auxins) and involved in induction of HSP and

sHSP promoters (Kotak et al. 2007; Carranco et al. 2010; Scharf et al. 2012). HSP101 codes for a

chaperone involved in protein remodelation through its ATPase activity (Singh and Grover 2010). This protein is not only implicated in Arabidopsis basal and acquired thermotolerance as it is regulated during seed development (Larkindale et al. 2007). HSP101 is accumulated during mid- maturation and stored in dry seed, in an expression pattern similar to that seen for LEA proteins and sHSPs (Xiong et al. 2001).

As expected, all stress-related transcripts were up-regulated in later stages of seed maturation (stages 5 and 6) (Figure 3.311A). In early stages of seed development (stages 1 and 2), only a

113 reduced expression was detected for Em6 and HSP101 genes. This could be due to the considerable expression of these genes in floral tissues as the samples harvested in the first stages of seed development contained remains of floral tissues (petals, stamens, pollen grains). Indeed, in

silico data (e-FP browser, BAR) predicted that HSP101 expression in carpels, stamens and petals. Em6 was only predicted to be up-regulated in later stages of seed maturation and dry and imbibed

(24 h) seeds. However, recent quantitative RT-PCR expression analysis data revealed that Em6 is ubiquitously expressed in different Arabidopsis organs, displaying highest levels in seedlings, buds and flowers (Hundertmark and Hincha 2008).

In the first germination day, seeds presented high Em1 and Em6 expression levels that significantly declined in the second day of germination (Figure 3.31B). During the germination process, the higher levels of Em transcripts, in relation to HSP101 and HSFA9, could be related with brief increased ABA levels during early phases of seed germination. This increase is crucial for environmental osmotic adaptation of germinating seeds, thus avoiding the damaging of important macromolecules. During germination, a much lower expression was observed for HSP101 and

HSFA9 coding genes. .

The expression analysis revealed significant expression differences between wild-type Ler and hrr mutant seeds. During seed maturation and in first day of germination, Em6 seems to be affected in developing hrr seeds (Figure 3.31A), while HSP101 and HSFA9 transcripts seem to be impaired in seeds during the first day of germination (Figure 3.31B). The expression impairment of these genes in hrr mutant suggests that HRR could be involved in the stability regulation of their transcripts. The expression regulation of Em6 has been proposed to be performed by ABI factors, through the interaction/modulation of ABI5 with ABI3 (Nakamura et al. 2001; Carles et al. 2002). Recent studies indicated that ABI3 could also activate the HSFA9 promoter, which in turn induces

HSP promoters, such as HSP101 (Kotak et al. 2007). In addition to a possible effect of HRR on

stabilisation of Em6, HSP101 and HSFA9 transcripts, the transcriptional network between ABI factors, HSFA9, HSP101 and Em proteins could enhance the HRR effect on seed development.

Far studying the relevance of HRR on transcription of Em1, Em6 and HSP101 genes, a semi- quantitative expression analysis was performed in imbibed seeds (wild-type Ler, hrr mutant and HRR over-expression lines), submitted or not to HS treatment (Figure 3.32). The expression profiles of Em genes were quite similar in wild-type Ler and hrr mutant seeds and in both experimental conditions. Only a slight reduction of HSP101 transcript levels was detected in hrr mutant seeds, in both experimental conditions. These results suggest that Em transcripts are not greatly affected by HS treatment (50ºC, during 60 min). Concerning the HRR over-expression lines, an up-regulation of all

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genes was detected, after HS treatment and also in control conditions. This result seems to be a good indication of the importance of high levels of HRR protein for the stabilisation of seed-related proteins.

Figure 3.32 Expression analysis of LEA protein genes (Em1, Em6) and HSP101 gene, in imbibed seeds, subjected or not to HS. Transcript levels were evaluated by semi-quantitative RT-PCR from mRNA extracted from imbibed wild-type Ler, hrr and HRR over-expression (JP9 and L2) mutant seeds, which were subjected to HS treatment (50ºC for 60 min) or were maintained at standard conditions (23ºC). As internal control, the transcript levels of Actin2 (Act2) were analysed. Numbers on the right correspond to the expected sizes of PCR products. The pair primers and PCR conditions are depicted in Annexes III and IV, respectively.

Altogether, the results showed that the evaluated stress-related genes (Em1, Em6, HSFA9 and HSP101) are preferentially induced in later stages of seed development (stages 5 and 6) and in early stages of seed germination. Almost all genes (Em6, HSP101 and HSFA9) appear to be regulated by HRR, once they were affected in hrr mutant in many of the developmental stages analysed. The up-regulation of Em1, Em6 and HSP101 in HRR over-expression line also supported this HRR role. The stability and integrity of stress-related transcripts would be crucial for seed development and germination, where dessication and osmotic stress conditions are stabilished.