LEA Gene Annotated As Being Similar to Heat-Shock Protein Precursor. (LOC Os12g32986;

Supplementary File 5: Written description of the functions of the predicted genes in the context of LEA gene function.

LEA gene annotated as being "Similar to Heat-shock protein precursor." (LOC_Os12g32986; Os12g0514500).

The LEA gene annotated as being "Similar to Heat-shock protein precursor." (LOC_Os12g32986; Os12g0514500) was found to share 17 common promoter motifs with a gene annotated as “Phosphatidylinositol 3- and 4-kinase, catalytic domain containing protein" (LOC_Os06g17290; Os06g0283400). These kinases are part of the Phosphatidylinositol (PI) signaling pathway in plants. This signaling pathway has been demonstrated to be critical in signal transduction processes such as cell differentiation, transduction of intracellular signaling molecules, control of cell responses to environmental factors, regulation of ion-channel gating, energy metabolism and rearrangement of cytoskeleton (Munnik et al., 1998). These kinases are responsible for the phosphorylation of intermediates in this pathway resulting in the generation of two important second messenger molecules, Ins(1,4,5)P3 and diacylglycerol (DAG), which can stimulate Ca2+ release from internal calcium stores and activate related protein kinases for regulation of downstream pathways or cellular responses.

The PI 3-kinase (PI3K) and PI 4-kinase (PI4K), synthesize PI 3-phosphate (PI3P) and PI

4-phosphate (PI4P) respectively. The plant PI3K has been suggested to be involved in root nodule development, plant growth and development, vesicle trafficking from Golgi to vacuoles, and regulation of the transcriptional process (Hong & Verma, 1994) (Welters et al., 1994; Bunney et al., 2000) (Kim et al., 2001a). Arabidopsis IP3K (AtIpk2α and AtIpk2β) is expressed in stem, leaf, stigma, siliques, and fast-growing regions including root tips and root hairs (Xia et al., 2003) (Xu et al., 2005), which implied that Arabidopsis IP3K may play important roles in plant growth and development. Mammalian IP3Ks are involved in a range of processes including brain development (Mailleux et al., 1991), embryogenesis (Frederick et al., 2005) memory and learning (Kim et al., 2004), membrane traffic and Ca2+ homoeostasis (Soriano et al., 1997) and oxidative stress resistance (Monnier et al., 2002).

PI4K catalyzes the production of PI4P, the only known precursor of PI45P, which can be cleaved into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol by phospholipase

C; therefore, it represents a critical point of regulation of PI-dependent pathways. In mammalian and yeast cells, PI4Ks also are important for membrane biogenesis and vesicle trafficking from the ER to the Golgi and the plasma membrane (Roth, 1999). In yeast, PI4K is required for the maintenance of vacuole morphology, cell wall integrity, and cytoskeletal organization, particularly during osmotic stress. In plant cells, two PI4K genes have been cloned (Stevenson et al., 2000). Previous studies have localized enzyme activity of these PI4Ks to the plasma membrane, nucleus, cytosol, and cytoskeleton (Drobak et al., 1999), their functions remain poorly understood. In Arabidopsis protoplasts knock outs in the AtPI4K gene resulted in a 50% reduction in Vesicular trafficking indicating that PPI species play a role in the turnover of intracellular membrane compartments (Kim et al., 2001a).

Phosphoinositide metabolism has been shown to play important roles in abscisic acid (ABA)–induced cytosolic calcium concentration changes and stomatal closing (Gilroy et al., 1990) (Staxen, I et al., 1999). In addition, endogenous PI monophosphate (PIP) levels, the product of the PI-kinases, were found to change rapidly in guard cells in response to treatment with ABA (Lee et al., 1996) and the products of the PI -3 and -4 kinases PI3P and PI4P were found to be involved in inducing ABA-induced stomatal closure in Vicia faba guard cells by increasing cytosolic Ca levels (Jung et al., 2002).

The LEA gene annotated as being "Similar to Heat-shock protein precursor." (LOC_Os12g32986; Os12g0514500) was also found to share 18 promoter motifs in common with the gene annotated as "Transcription factor jumonji, JmjN domain containing protein" (LOC_Os05g10770; Os05g0196500). The jumonji (jmj) gene was initially identified using a mouse gene trap approach and is characterized as essentual nuclear factor involved in mouse embryonic development (Takeuchi, 1997). Jmj has been shown to play important roles in cardiovascular development, neural tube fusion processes, hematopoiesis, and liver development in mouse embryos (Jung et al., 2005). In many diverse species including bacteria, fungi, plants, and animals, there are many jumonji family proteins. Recently, Jmj protein was found to be a transcriptional repressor. Several proteins in the jumonji family are involved in transcriptional repression and/or chromatin regulation (Takeuchi et al., 2006).

Interesting, along with a number of LEA and heat shock encoding genes, a Jumunji class transcription factor was present in a water stress deficit cDNA library in the peanut plant. In the peanut plant the expression of a jumunji transcription factor protein was determined to be upregulated in response to water deficit and the expression of the homolog of this protein was also found to be induced in water deprived wildtype N. benthamiana plants. Additionally, inhibition of expression of this Jumonji protein by virus-induced gene silencing (VIGS) resulted in increased membrane damage following water deprivation in Nicotiana benthamiana (Senthil-Kumar et al., 2007).

The sequence, secondary-structure and active-site similarities between JmjC domains and cupins strongly indicate that JmjC domains represent a previously unrecognized branch of the cupin family with mostly enzymatic functions (Clissold & Ponting, 2001). Similarities between cupins and several transcription factors had previously been noted and wer subsequently identified to contain JmjC domains, such as rat testis-specific protein A (a hairless paralogue) and a DNA-binding protein (ENBP1) from Vicia sativa (Clissold & Ponting, 2001) (Dunwell, 1998).

This LEA gene also shared 17 promoter motifs with “Ribulose bisphosphate carboxylase, small chain family protein” (LOC_Os11g19250.1; Os11g0298400). Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant protein in nature and is the primary enzyme in photosynthetic carbon fixation and the likely rate limiting factor for photosynthesis under light saturated conditions and atmospheric CO2 pressures (Makino et al., 1985). In the monocot Hordeum vulgare (barley) protein levels of the small subunit of Rubisco was found to decrease in abundance during seed development and maturation (Finnie et al., 2002) and in rice seedlings ABA has been shown to reduce rubisco levels in a time dependent manner (Rakwal & Komatsu, 2004). Further, in rice leaves mRNA transcripts and the syntheis of rubisco subunits was drastically reduced in response to cold stress (Hahn & Walbot, 1989). Oxidative conditions have been shown to cause fragmentation and degredation of Rubisco (Penarrubia & Moreno, 1990) (Ishida et al., 1999). It has been suggested that ROS may modify Rubisco sulfhydryl groups from critical cysteine residues whose oxidation renders the enzyme sensitive to proteases (Penarrubia & Moreno, 1990) (Moreno et al., 1995). Further, in tobacco, both a decrease in the levels of mRNA encoding the small subunit of Rubisco (Kawaguchi R, 2003), and a decreased amount of Rubisco protein and have been observed in response to water stress conditions.

Abscisic acid (ABA) treatment and water stress have been commonly reported to induce stomatal closure and an inhibition of leaf photosynthesis (Downton et al., 1985) (Terashima et al., 1988). Evidence indicates that stomatal closure is responsible for the reduction in photosynthesis following water stress due to the limited CO2 diffusion from the atmosphere to the site of carboxylation (Chaves & Oliveira, 2004) (Flexas et al., 2004) .

The decline in intracellular CO2 levels results in the over-reduction of components within

the electron transport chain and the electrons get transferred to oxygen at photosystem I (PS I). This generates ROS including superoxide, hydrogen peroxide (H2O2) and

hydroxyl radicals (Lorento et al., 1995). Plants need to respond quickly to prevent the photosynthetic machinery from suffering from irreversible oxidative damage. Since most studies on drought-induced changes indicate that the amount and activity of rubisco primarily controls photosynthetic carbon assimilation, the reduction in rubisco levels may contribute to the shut down of photosynthesis and thus help prevent the generation of ROS and damage to the photosynthetic machinery during water deprivation.

This LEA gene was also found to share 17 promoter motifs with a gene annotated as "Peptidase A1, pepsin family protein" (LOC_Os07g34920.1; Os07g0533600). Aspartic proteinases (APs ) have been extensively studied and characterized and are widely distributed among vertebrates, plants, yeast, nematodes, parasites, fungi and viruses (Davies, 1990) (Dunn, 2002). Plant APs are widely distributed in the plant kingdom and have been extensively detected in monocotyledonous (Asakura et al., 1995) (Sarkkinen et al., 1992) and dicotyledonous (Hiraiwa et al., 1997) (Mutlu et al., 1998) plants. The majority of plant APs belongs to the A1 family. In common with other members of the A1 family, plant APs are active at acidic pH, are specifically inhibited by pepstatin and have two aspartic acid residues responsible for the catalytic activity (Dunn, 2002). In situ hybridization demonstrated that A1-protease genes are expressed in many cells of the seeds and developing seed pods.

Plant APs have been implicated in general protein processing and/or degradation in numerous organs and processes including senescence, stress responses, programmed cell death and reproduction (Simoes & Faro, 2004). In situ hybridization demonstrated that A1-protease genes are expressed in many cells of the seeds and developing seed pods.

Participation of plant APs in storage protein degradation during the mobilization of reserve proteins in seed germination has been proposed for rice and wheat since the distribution of APs was co-localised with seed storage proteins (Doi et al., 1980). Additionally, AP was able to hydrolyse the main wheat storage protein gliadin (Belozersky et al., 1989). Based on experimental evidence a role for plant APs in proteolytic processing and maturation of seed storage proteins has also been proposed (Mutlu et al., 1998; Runeberg-Roos et al., 1994).

This LEA gene was also found to share 17 promoter motifs with a gene annotated as "DEAD/DEAH box helicase N-terminal domain containing protein” (LOC_Os07g43980.1; Os07g0633500). DNA repair genes are included among genes that are induced by abiotic stresses since these types of stresses often result in DNA damage. Since the stresses affect the cellular gene expression machinery, it is possible that molecules involved in nucleic acid metabolism including helicases are likely to be affected (Vashisht & Tuteja, 2006). Most helicases are members of DEAD-box protein superfamily and play essential roles in basic cellular processes such as DNA replication, repair, recombination, transcription, ribosome biogenesis and translation initiation (Matson et al., 1994) (West, 1996). It was proposed that helicases might play an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways (Vashisht & Tuteja, 2006).

There are reports of abiotic induced induction of DEAD-box helicases in a number of plant species (Vashisht & Tuteja, 2006). In barley a salt responsive transcript HVD1 (Hordeum vulgare DEAD-box protein), encoding a putative ATP-dependent DEAD-box RNA helicase was reported to be increased under salt stress, cold stress and ABA treatment (Nakamura et al., 2004). In Arabidosis plantsa a DEAD-box RNA helicase was identified to be expressed during cold stress (Seki et al., 2001). In Pea a DNA helicase 45 (PDH45), was found to be induced in pea seedling in response to high salt (NaCl) and other abiotic stresses including dehydration, wounding and low temperature (Sanan-Mishra et al., 2005). The induction of PDH45 transcript was observed to be induced by ABA, which suggested that the stress effect may be mediated through ABA-mediated pathways.

LEA gene “Similar to Dehydrin DHN1 (M3) (RAB-17 protein)” (LOC_Os11g26570.1; Os11g0451700).

The LEA gene annotated to be “Similar to Dehydrin DHN1 (M3) (RAB-17 protein)” was found to share 9 promoter motifs with a gene annotated as being “Similar to Heat shock protein STI (Stress inducible protein) (GmSTI)" (LOC_Os02g43020.1; Os02g0644100) that contains tetratricopeptide regions (TPR).

This gene was first identified in yeast where its transcript levels were determined to be induced by heat shock (named STI 1). Additionally, the STI gene product is required for optimal growth of yeast cells at both low and high temperatures (Nicolet & Craig, 1989). In soybean, a protein named GmSTI was found too share high sequence identity to the yeast STI1 stress-inducible protein which has a TPR elements (Hernandez et al., 1995). The GmSTI gene was found to be heat-inducible and was the first evidence for the existence of plant genes coding for proteins which belong to the TPR family. STI1, IEF SSP 3521 and GMSTI all contain a number of 34-aa TPR which are proposed to be important for intra- and intermolecular protein interactions (Lamb et al., 1995). In yeast and humans both STI1 and IEF SSP 3521 have been identified as components of multiprotein chaperone complexes involving the heat shock proteins, Hsp70 and Hsp90 (Smith et al., 1993) (Chang & Lindquist, 1994) (Schumacher et al., 1994) and evidence indicates that STI may play a role in mediating the heat shock response of some HSP70 genes (Nicolet & Craig, 1989). The STI proteins are also referred to as Hop (hsp70- and hsp90-organizing protein) based on their functions as adapter or cochaperone proteins that can bind to both hsp90 and hsp70 simultaneously, bringing them into close proximity (Chen & Smith, 1998). The Arabidopsis genome sequence has been examined for Hop-like sequences, and three genes encoding Hop-like proteins were identified (Krishna & Gloor, 2001), confirming the presence of a multigene family for this protein in plants. The HSP proteins are a stereotypical set of proteins that have been shown to be synthesized in response to increased temperature in cells from nearly every organism thus far tested (Nicolet & Craig, 1989). The synthesis of these proteins is also induced by a number of other stress conditions indicating that they are involved in general cellular stress responses. The most abundant and highly conserved of the heat shock proteins found after a temperature increase is called hsp70. Analysis of thermoregulation of HSP70 genes, and of other heat shock genes from a number of organisms, has led to the hypothesis that the primary function of the heat shock proteins is to protect cells from damage caused by an abrupt shift in environmental conditions. Hsp70 protects cells from stress by binding partially unfolded proteins preventing protein aggregation and prion formation (Tutar, 2006). Thus, the identification of a stress inducible cochaperone to share common promoter motifs with a stress inducible LEA gene is consistent with these products being co-regulated since they are both involved in protecting cellular components when adverse conditions are encountered.