Diseño del eje soportante de la cabina

Seasonal changes in ryegrass gene expression in samples collected from active paddocks during

different seasons were analysed using SAGETM _{(Serial Analysis of Gene Expression) based ryegrass}

transcriptome program (Sathish et al., 2007). This SAGE based gene expression analysis facilitated by a proprietary GeneThresher® database (methyl filtered sequence mapping) (Bajaj et al., 2007) resulted in identification of LpUbl5. LpUbl5 gene expression was increased about three fold under water deficit when compared with before stress, rehydration and summer heat condition. This indicated that LpUbl5 may have a potential role in drought response in ryegrass. LpUbl5 was over-expressed in ‘pasture type’ perennial ryegrass ‘Grasslands Impact’ NZ Agriseeds, Christchurch, NZ using vector pLpHUB1 in which a Double CaMV35S (D35S) promoter driving LpUbl5 and another D35S driving the expression of marker gene, hptII for hygromycin resistance. ‘Turf type’ perennial ryegrass ‘Monterey II’ (MT) was transformed using pTTS44 with 35S promoter driving the LpUbl5 gene and another 35S promoter driving the bar gene (bialaphos resistance) encodes phosphinothricin acetyl transferase (PAT) (Patel et al., 2015). Embryogenic calli derived from meristematic regions of the vegetative tillers were transformed using high throughput Agrobacterium-mediated genetic transformation (Bajaj et al., 2006). Patel et al. (2015) reported higher shoot biomass accumulation, increased relative water content, reduced water potential and increased chlorophyll content under stress when compared with controls. A two to three fold increase in the LpUbl5 transcript level was observed under drought when compared with the transcript level before imposing drought stress in vector only control and transgenic lines. This has been promoted as evidence for its involvement in enhanced drought tolerance (Patel et al., 2015). However, their study did not identify a physiological response as a result of the over-expression of LpUbl5 to support the mechanistic role of LpUbl5 in plants under drought stress conditions. Therefore, evaluation of physiological response under imposed drought help to

investigate its hypothesised role in drought response. This led to the present evaluation of T3

generation of ‘pasture type’ perennial ryegrass to further evaluate and understand the physiological functions of Ubl5 in ryegrass as highlighted in Figure 2-1. The research reported in this thesis is focused on the physiology measurements and transcript abundance of LpUbl5 in ryegrass under imposed drought cycles in controlled environment. Further, efforts were taken to evaluate the function of Ubl5 in Arabidopsis, as a model plant under drought, and also incorporated into Nicotiana Benthamiana and Allium Cepa L. to investigate the subcellular localization of LpUBL5.

23

2.5

Arabidopsis as a model plant

Arabidopsis thaliana (family Brassicaceae) has been used in experimental research for about half a century (Page and Grossniklaus, 2002). Arabidopsis was the third multicellular organism, and first plant to be sequenced, known to have the smallest plant genome (125 Mb) (Initiative, 2000; Bevan and Walsh, 2005). A. thaliana was designated as a model plant in 1943 due to its small plant size, rapid generation within 5-6 weeks under optimum conditions, ability to grow in soil or defined media under controlled conditions, ability to generate up to 10,000 seeds per plant via self-pollination and easy maintenance (Page and Grossniklaus, 2002). Apart from these key features, efficient Agrobacterium mediated transformation of Arabidopsis allowed researchers to express or over express the gene of interest and analyse the effect of newly introduced genes within short time periods due to its short life cycle. These are also highly efficient methods to generate insertional mutants such as agrobacterial plasmid T-DNA transformation and transposon based mutants. Subsequently, high-throughput ways of sequencing simplified the laborious stage in reverse genetics. The availability of these mutant seed stocks of any gene of interest from publically available resources has simplified gene function studies. Researchers are able to study gene function in Arabidopsis mutant lines which helps to identify the functions of an orthologous gene of interest in other plants species (Provart et al., 2016).

Arabidopsis was incorporated in drought tolerance studies either by genetically modifying the plant using the gene of interest or by screening the mutant lines of specific genes. Over expression of genes such as, ABRE binding factors 3 and 4 (ABF3/4) (Kang et al., 2002), AtMYC2/AtMYB2 (Abe et al., 2003), CBF4 (Haake et al., 2002), SRK2C (Umezawa et al., 2004), HDG11 (Yu et al., 2008), XERICO (Ko et al., 2006), MYB15 (Ding et al., 2009), ADC2 (Alcázar et al., 2010), DREB1A and DREB2A (Liu et al., 1998) have successfully demonstrated enhanced drought tolerance in Arabidopsis. Incorporation of mutant lines to study drought sensitivity using genes such as max2 (MAX2 gene) (Bu et al., 2014), study of SIZ1 using siz1-3 mutants and a complementation line C-siz1-3 (Catala et al., 2007) and ABO3 using abo3 mutant line and Line 4 (ABO3 overexpressing in abo3 mutant line) (Ren et al., 2010) are examples of utilizing mutant lines to query gene function. Incorporation of both mutants and overexpression of the gene of interest, to identify drought responsive genes, CAMTA 1 (Pandey et al., 2013), and YUC6 (Cha et al., 2015) , WRKY57 (Jiang et al., 2012) are also a method of choice.

2.5.1

Arabidopsis thaliana Ubl5 gene (AtUbl5)

Plant response to drought or water deficit can be measured from the whole plant to the molecular level. Technologies such as micro arrays help to understand expression patterns of different genes simultaneously and allows us to understand the types and quantities of RNA in a cell in response to drought (Bray, 2004). A review of literature suggests several studies have been published to date to identify the molecular response of Arabidopsis under different abiotic stresses such as drought, heat, salt and UV radiation (Seki et al., 2001; Kreps et al., 2002; Seki et al., 2002; Oono et al., 2003; Kilian et

24 al., 2007; Huang et al., 2008). The gene of interest of this study, Ubl5 (At5g42300) was looked up in published literature to identify any involvement of Ubl5 in response to drought and ABA. At5g42300 was identified as drought induced, ABA and PBI425 (ABA analogue) induced gene out of 1310 other genes. The comparison of data was between results obtained from water withdrawn and rehydrated aerial parts of the plants (Huang et al., 2008). Whereas, a former study by the same authors listed At5g42300 as a gene induced by PBI425 at 24 hours along with 1501 genes whereas the same study did not list this gene under ABA (20 µM) induced either at 6 or 24 h (Huang et al., 2007). This variation was explained as PBI425 in high concentration and long persistence contributing to the expression of more ABA-responsive genes and thereby increasing drought tolerance (Huang et al., 2008). However, a meta-analysis using the data generated by Huang et al. (2008) did not list At5g42300 in differentially expressed gene (DEGs) under drought and re-watering (Xu and Wu, 2013). Apart from this, a challenge to these above results was found in study by Wang et al. (2011) where 30 µM ABA treated plants were used for analysis. At5g42300 was detected in both leaves and guard cells of the plants but they did not list At5g42300 as an ABA-responsive gene (Wang et al., 2011). Li et al. (2006) studied ABA-responsive (10 µM) genes in Arabidopsis and did not list At5g42300 as an ABA-responsive gene. Furthermore, 2678 Arabidopsis genes were listed as genes which showed a 2 fold change in response to drought in 3 week old seedling where the drought was initiated by transferring plants to 200 mM mannitol in hydroponic medium. These did not include At5g42300 (Kreps et al., 2002). Similarly, studies which queried genes under drought using either soil grown of hydroponic medium grown plants did not list At5g42300 as a drought induced gene (Seki et al., 2002; Oono et al., 2003; Kawaguchi et al., 2004; Kilian et al., 2007). In contrast, another group of studies have reported both At5g42300 and At3g45180 as drought responsive genes. At3g45180 was upregulated by drought after 5 days, and continued to be upregulated at 7 and 9 days of water withdrawal in shoots. Whereas, At5g42300 was reported to be upregulated only by 9 days after water withdrawal, when compared with 0 days. This study used 17 day old soil grown plants subjected to water withdrawal up to 9 days and samples were collected on 0, 1, 3, 5,7 and 9 days. At3g45180 and at5g42300 were not up-regulated in root (Rasheed et al., 2016). Two week old media grown plants subjected to dehydration by leaving the harvested plants on parafilm have reported At3g45180 (1 fold) and At5g42300 (2.9 fold) changes under drought, when compared as droughted versus wild type well-watered (Nishiyama et al., 2013). In other study, plants were grown in pellets and moderate (30% of the field capacity) or progressive drought (complete water withdrawn) drought were applied to 35 day old plants. At5g42300 was upregulated only by progressive drought (until symptoms of wilting was observed) and not by ABA treatments or moderate drought (Harb et al., 2010). In a separate study using soil grown 3 week old plants, At3g45180 (-1.5) was down regulated and At5g42300 (1.4 fold) was upregulated in wild type droughted plants versus wild type well watered plants (Van Ha et al., 2014). These studies differ in

25 terms of their plant growth medium, application of drought/water deficit conditions, time of exposure of plants to stress, type of samples employed and their controls to which they compare the data to identify the fold change. Collectively results are inconclusive so Ubl5 (At3545180 or At5g42300) cannot yet be designated as a drought responsive gene.