aDepartment of Utilized Organic Chemistry, Graduate Faculty of Agricultural and Life Sciences, The College of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
bPlant Science Middle, The Institute of Bodily and Chemical Analysis (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
bPlant Science Middle, The Institute of Bodily and Chemical Analysis (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
cDivision of Utilized Bioscience, Graduate Faculty of Agriculture, Hokkaido College, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
cDivision of Utilized Bioscience, Graduate Faculty of Agriculture, Hokkaido College, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
dCorresponding writer:
pj.ca.iadukoh.rga.sba@otian; fax 81-11-706-4932; cellphone: +81-11-706-2800
Summary
Introduction
Plant storage proteins may be labeled into two courses; seed storage proteins (SSPs) and vegetative storage proteins (VSPs). SSPs are a set of proteins that accumulate to excessive ranges in seeds through the late phases of seed growth, whereas VSPs are proteins that accumulate in vegetative tissues resembling leaves, stems and, relying on plant species, tubers. Throughout seed germination, SSPs are degraded and the ensuing amino acids are utilized by the creating seedlings as a dietary supply. SSPs are the key proteins in grains and, of the plant proteins, symbolize these which are probably the most abundantly consumed by people. Then again, VSPs accumulate within the vegetative tissues when extra assets can be found and function a temporal reservoir of amino acids to be used in subsequent phases of progress and growth.
SSP genes are traditional targets for plant molecular biology. Their plentiful expression in seeds allowed for straightforward detection of the gene transcripts and cDNA cloning through the daybreak of plant molecular biology in late 70’s to early 80’s. Their expression is strictly restricted to creating seeds through the mid- to late-stages of embryogenesis, a attribute that makes them supreme for the research of tissue- and temporal-specific regulation of gene expression. Research on the expression of SSP genes has contributed tremendously to the event of plant molecular biology. Molecular genetic research utilizing Arabidopsis have led to an additional understanding of expression and accumulation of SSPs and the current genomic exploration of Arabidopsis has additionally begun to influence on our understanding of seed maturation processes related to SSP accumulation.
On this overview, we’ll summarize the research on the regulation of SSP accumulation in Arabidopsis as regards to different seed-specific genes. The VSPs of Arabidopsis are additionally described by way of genomic group and regulation of gene expression.
Seed storage protein genes of Arabidopsis
The foremost SSPs of Arabidopsis are comprised of 12S globulins and 2S albumins (Determine 1). This classification of SSPs as albumins or globulins relies on whether or not the proteins are water-soluble or soluble in salt answer, respectively. Along with this classification, the SSPs of crops additionally usually have particular names. For instance, glycinin, glutelin and zein symbolize particular SSP fractions from soybean, rice and maize, respectively. The Arabidopsis SSPs have additionally been named after these of Brassica napus. 12S globulin proteins are known as cruciferin, whereas the 2S albumins are known as both napin or arabin.
Each 12S globulins and 2S albumins are initially synthesized as a precursor and accumulate in protein our bodies after processing of the preproteins. The 12S proteins are a member of the legumin-like 11S globulins and accumulate as hexameric complexes consisting of six a and 6 b polypeptides linked through disulfide bridges. The a and b polypeptides are generated from a single polypeptide-precursor following particular cleavages by processing enzymes. Cysteine residues concerned within the crosslinking of the polypeptides and formation of the hexameric complexes have been recognized (Inquello et al., 1993). In distinction, 2S albumin accumulates as a heterodimer consisting of two subunits linked by disulfide bridges. Just like that for 12S globulin, these subunits are generated by cleavage of a precursor polypeptide (Krebbers et al., 1988). Synthesis of each 12S and 2S SSPs takes place on the tough ER, adopted by sorting into protein our bodies.
Just like that in most different plant species, Arabidopsis SSPs are encoded by a small multi-gene household (Desk 1). 5 isoforms of genes encoding 2S albumin, known as at2S1 to at2S5, have been reported (Krebbers et al., 1988; van der Klei et al., 1993). Of those, 4 of the 2S albumin genes (at2S1 by way of at2S4) are current as a tandem cluster within the decrease half of chromosome 4 (approx 12,575 kbp place). The fifth gene of 2S albumin (At5g54740; van der Klei et al., 1993) is positioned within the decrease area of chromosome 5. Proteins similar to all of those genes have been detected, suggesting that every one 5 genes are actively transcribed (van der Klei et al., 1993). Phylogenetic evaluation of the amino acid and nucleotide sequences of 2S albumin genes in Brassicaceae means that duplication of the 2S albumin genes occurred previous to the Brassicaceae-Sysimbrieae break up adopted by concerted evolution (Boutilier et al., 1999).
Cloning of the 12S globulin genes of Arabidopsis was first reported by Pang et al. (1988). Three genes encoding 12S globulin are current within the genome of Columbia ecotype. These genes, designated as CRA1, CRB and CRC, are positioned on chromosome 5, 1 and 4, respectively (Desk 1). The Landsberg erecta genome comprises one other copy of the 12S globulin gene, CRA2, which is positioned in tandem with CRA1 (Pang et al., 1988).
Apart from the 12S and 2S proteins, 7S globulin proteins (vicilins) are additionally among the many main SSPs of many plant species. Arabidopsis doesn’t comprise detectable 7S storage protein in mature seeds however a vicilin-like gene (At2g18540) is current in direction of the center of chromosome 2 in a area lined by the BAC clone T17D12. A search of the Arabidopsis genome database has additionally revealed two different globulin-like genes, one within the higher area of chromosome 1 (At1g07750, BAC clone F24B9) and the opposite close to the underside of chromosome 4 (At4g36700, BAC clone AP22). A lot of ESTs from siliques or seeds match At1g07750 or At4g36700, suggesting that these genes are expressed in seeds or siliques.
A lot of genes encoding proteins apart from SSPs are additionally expressed abundantly throughout late embryogenesis. These embrace oleosins and late embryogenesis plentiful (LEA) proteins. Oleosins are comparatively small hydrophobic proteins (15-25 kD) related to oil our bodies, a mobile organelle for storage of triacylglycerols. They’re synthesized particularly in seeds and thought of to be essential for stability of oil our bodies. LEA genes are a set of genes which are expressed in later phases of embryogenesis. LEA genes encode proteins which are extremely hydrophilic and are proposed to play a task within the acquisition of desiccation tolerance. The expression of LEA genes is nonetheless not all the time strictly restricted to late embryogenesis and plenty of LEA genes are additionally expressed vegetatively in response to numerous stresses or in reproductive tissues, resembling pollen. Two different genes, ATS1 and ATS3 (Arabidopsis
thaliana
seed genes), have additionally been recognized in Arabidopsis as seed-specific genes that have been remoted by way of a differential show methodology (Nuccio and Thomas, 1999). Then again, AtEm1 and AtEm6 are expressed in each late embryogenesis and vegetative tissues in response to exogenous utility of abscisic acid (ABA) (Soderman et al., 2000). Current progress in genomic research utilizing Arabidopsis has tremendously elevated the variety of seed-specific genes recognized (Girke et al., 2000).
Expression of seed storage protein genes — cis-acting components
Just like different SSP genes, the SSP genes of Arabidopsis are expressed particularly through the mid- to late-stages of seed growth. Nevertheless, the detailed patterns of expression throughout these developmental phases do differ between the genes.
The at2S1 gene is expressed basically within the embryo axis, whereas at2S2 and at2S3 genes are extremely expressed all through the embryo (Guerche et al., 1990). An identical distinction in expression ranges and patterns has additionally been noticed in corresponding promoter-reporter constructs, suggesting that the expression ranges and tissue specificity are transcriptionally regulated (Conceicao et al., 1994). Research utilizing hybrid promoter constructs between at2S1 and at2S2 recognized a area of the promoters required for 2S albumin expression in palisade parenchyma and particular epidermal cells (Conceicao and Krebbers, 1994). It was additionally demonstrated that a number of the hybrid promoters exhibited altered temporal regulation (Conceicao and Krebbers, 1994).
5′-deletion evaluation of the at2S1 promoter outlined a area required for seed-specific expression (Conceicao and Krebbers, 1994), though no additional evaluation of cis-acting components chargeable for seed-specific expression of Arabidopsis SSPs has been reported to this point. Detailed promoter evaluation has been largely carried out in legumes and cereals (Shewry et al., 1995; Wohlfarth et al., 1998 for overview). These analyses recognized the RY repeat motif (additionally refered to because the Sph component or legumin field; Baumlein et al., 1992; Chamberland et al., 1992; Fujiwara and Beachy, 1994; Sakata et al., 1997; Bobb et al., 1997) as a key cis-acting component for seed-specific gene expression. The RY repeat is a extremely conserved cis-acting component appearing as each an enhancer for seed-specific gene expression and a repressor of expression in vegetative tissue. A sequence similar to the RY repeat motif or a sequence associated to it’s also current in promoter areas of Arabidopsis SSP genes (Conceicao and Krebbers, 1994).
Evaluation of cis-acting components of the Arabidopsis oleosin promoter in transgenic B. napus revealed DNA fragments concerned in seed-specific gene expression (Plant et al., 1994). Sequences current between −2500 bp and −1100 bp of the promoter have been proven to be concerned in modulating the degrees of gene expression through the early phases of embryo growth, whereas the −400 bp to −200 bp area contained sequences concerned in osmotic induction. Comparable promoter deletion analyses have additionally been reported for different Arabidopsis genes expressing within the seed, together with LEA genes (Hull et al., 1996).
Regulation of seed storage protein gene expression — trans-acting components – “where is protein stored”
Genetic research have been important in elucidating the molecular mechanisms underlying regulation of SSP gene expression. Mutations inflicting reductions in SSP ranges are highly effective instruments within the effort to grasp the community of transcriptional regulation of SSP genes. There are two recognized examples demonstrating an interplay between a cis-acting component and its trans-acting issue within the regulation of SSP gene expression in Arabidopsis. These are the RY repeat/B3 area proteins (ABI3 and FUS3) and the ABA-responsive component (ABRE)/bZIP protein (ABI5). Different loci affecting the expression of SSP genes seem to behave both through one other cis component(s) controlling SSP gene expression or not directly by modulating “direct” trans-acting components.
The abi3 (abscisic acid-insensitive3), fus3 (fusca3), and lec1 (leafy cotyledon1) mutants produce seeds which are illiberal to desiccation (Nambara et al., 1992; Keith et al., 1994; Meinke et al., 1994; West et al., 1994). When these mutant seeds are harvested and sown on medium previous to desiccation they present regular progress, though desiccation illiberal seeds are nonetheless produced within the subsequent era. Accumulation of SSPs and LEA proteins is severely diminished in these mutants (Nambara et al., 1992; Vicient et al., 2000) and the profile of the worldwide sample of gene expression can be altered throughout mid- to late-embryogenesis. Throughout these phases, SSP and LEA genes are downregulated and genes usually expressed throughout or after germination are precociously activated (Nambara et al., 2000; Parcy et al., 1994, 1997; West et al., 1994). Due to this fact, ABI3, FUS3, and LEC1 seem to behave as essential regulatory parts of mid- to late-stages of embryogenesis. One other class of mutants additionally exists that exhibit altered expression of LEA genes, though they’ll produce desiccation tolerant seeds. The abi4 (abscisic acid-insensitive4) and abi5 (abscisic acid-insensitive5) mutants are faulty in gene expression of a subset of LEA genes throughout late-embryogenesis (Finkelstein, 1994). Though the monogenic mutants of those genes exhibit little or no phenotype in SSP gene expression, different information suggests ABI4 and ABI5 are concerned in regulation of SSP gene expression. The ABI3, FUS3, LEC1 and ABI4 trans-acting components are mentioned individually under with respect to their function in regulation of seed storage proteins.
The ABI3 gene encodes a transcription issue, which is an ortholog of the maize VP1 gene (Giraudat et al., 1992). The phenotype of maize vp1 mutants is sort of just like that of abi3 mutants that produce desiccation illiberal ABA-insensitive seeds (McCarty et al., 1991). ABI3/VP1 orthologs from each monocots and dicots comprise 4 conserved domains (A, B1, B2, and B3; Determine 2). The B3 area of VP1 is ready to bind particularly to the Sph component that comprises a RY repeat in vitro (Suzuki et al., 1997) and it has been established that the RY repeat is required for transcriptional activation by ABI3 (Reidt et al., 2000). Then again, involvement of the B2 area of ABI3 in regulation of At2S2, AtEm1, and AtEm6 gene expression is recommended (Bies-Etheve et al., 1999). Moreover, ABI3 seems to behave in live performance with different transcription components. Transcription components interacting with ABI3 have been recognized and are recommended to be a element of the transcriptional regulatory community for embryo growth in Arabidopsis (Kurup et al., 2000).
The ABI3 gene is expressed abundantly throughout embryogenesis, which ceases after germination (Parcy et al., 1994). Current reviews have demonstrated that it’s also expressed in dormant tissue, thus suggesting one other operate of ABI3 in tissues apart from seeds (Rohde et al., 2000, for overview). Transgenic Arabidopsis carrying a CaMV 35S promoter (35S):ABI3 assemble expresses a number of the SSP genes ectopically in response to exogenous ABA, demonstrating that ABI3 is without doubt one of the important components for seed-specific gene expression (Parcy et al., 1994).
The FUS3 gene encodes a B3 domain-containing transcription issue that lacks the N-terminal sequence, a conserved sequence in different orthologs of ABI3/VP1 (Luerssen et al., 1998) (Determine 2). The RY repeat has additionally been proven to be below the regulation of FUS3-mediated transcriptional activation (Wohlfarth et al., 1998, for overview) and it was lately demonstrated that the RY repeat is the direct goal of the B3 area of FUS3 (Reidt et al., 2000). Lately it was demonstrated that the LEC2 gene, whose mutant reveals related phenotype as does the fus3 mutant however reveals partial intolerance to desiccation, additionally encodes a B3 domain-containing protein (Stone et al. 2001). Different transcription components containing the extremely conserved B3 area of VP1/ABI3/FUS3, designated as RAV1 and RAV2, are reported (Kagaya et al., 1999).
Though ABI3 and FUS3 act as transcriptional regulators cooperatively through the RY repeat, in addition they operate independently to manage SSP gene expression. This implies that different domains are additionally essential in defining their distinct capabilities. One of many essential variations between ABI3 and FUS3 is the distinct function within the ABA response in seeds. The rice bZIP transcription issue TRAB1 that binds to ABRE interacts with the N-terminal area of OsVP1, a rice VP1 ortholog, in an ABA-dependent method (Hobo et al., 1999). Curiously, the Arabidopsis ABI5 gene encodes a bZIP transcription issue that’s just like Rice TRAB1 (Finkelstein and Lynch, 2000) and it additionally seems to bind to ABRE in vitro (Lopez-Molina et al., 2001). The N-terminal area of ABI3 (A and B1 domains), which is lacking in FUS3, seems to be essential for ABA-induced transcriptional activation by ABI3 (Reidt et al., 2000). Moreover, ABI5 is required for the activation of SSP genes in vegetative tissues by 35S:ABI3 (Soderman et al., 2000). Lately Nakamura et al. (2001) reported that the B1 area of ABI3 is important for interplay with ABI5 in a yeast two-hybrid assay.
The LEC1 gene is expressed in a seed-specific method and encodes a putative transcription issue that could be a homolog of the subunit of CAAT box-binding issue (Lotan et al., 1998), though the goal DNA sequence of LEC1 is but to be recognized. Just like that for the fus3 mutant, trichomes are current on leaf cotyledons of the lec1 mutant, suggesting a metamorphosis of cell-type specificity (Determine 3). Ectopic expression of the LEC1 gene ends in progress arrest after germination and infrequently produces embryo-like buildings expressing SSP genes in these organs. This means that expression of the LEC1 gene is adequate to provoke embryo growth (Lotan et al., 1998). Curiously, At2S1 and oleosin genes are ectopically expressed in roots of the pickle (pkl) mutant, which misexpresses the LEC1 gene in these tissues (Ogas et al., 1997, 1999). The PKL gene encodes a chromatin-remodelling issue (Ogas et al., 1999), suggesting that PKL-mediated repression of the LEC1 gene is essential to repress seed-specific gene expression in non-embryonic tissues.
Expression of 2S albumin genes can be depending on genome group. As talked about above, 4 genes encoding 2S albumin are clustered throughout the Arabidopsis genome. Steady introduction of a transgene carrying a tandem repeat of 2S albumin genes into Arabidopsis resulted in unexpectedly excessive expression ranges of the assemble (Conceicao et al., 1994), suggesting an significance of gene group in regulation of the 2S albumin genes. Chromatin construction has additionally been proven to be concerned in seed-specific expression of the phaseolin gene in French bean (Li et al., 2001, for overview). In vivo footprinting experiments of the phaseolin gene promoter revealed that nucleosome positioning is altered in seed to permit entry of transcriptional regulators. The transcription issue PvALF, a French bean ortholog of ABI3/VP1, is intrinsically concerned in nucleosome positioning, as are different non-histone proteins and ABA.
The ABI4 gene encodes a transcription issue containing an AP2-domain, a conserved DNA-binding area in vegetation (Finkelstein et al., 1998). The goal sequence of ABI4 stays unidentified, though the construction of the DNA binding area is just like that of ethylene responsive component binding protein or the cold-responsive component binding protein, CBP1. Expression of the ABI4 gene is plentiful throughout embryogenesis, and it’s also weakly expressed in vegetative tissues. A few of the genes encoding LEA proteins (AtEm1, AtEm6, and PAP140) have additionally been discovered to be downregulated in abi4 mutant seeds (Finkelstein et al., 1998). Though loss-of-function of the ABI4 gene doesn’t considerably have an effect on the expression of 12S globulin nor 2S albumin genes, transgenic vegetation carrying the 35S:ABI4 assemble are capable of ectopically categorical the SSP genes together with At2S3 and CRC genes in response to exogenous ABA.
Genomic research on seed-specific gene regulation
As a part of a research on lipid metabolism in seeds, micro-arrays containing roughly 5,000 seed-expressed Arabidopsis genes have been used to additional perceive seed-specific gene expression. Comparability with probes derived from seeds, leaves and roots revealed that roughly 10% of the genes have been expressed at ratios higher than or equal to 10-fold greater in seeds than in leaves or roots. Included on this listing are numerous proteins of unknown operate, and potential regulatory components resembling protein kinases, phosphatases and transcription components (Ohlrogge et al., 2000). These components could also be concerned in seed-specific regulation of gene expression. In one other research utilizing micro-arrays containing roughly 2,600 seed-expressed genes, roughly 25% of the genes have been expressed at ratios higher than or equal to 2-fold greater in seeds than leaves or roots and 10% at ratios higher than or equal to 10 (Girke et al., 2000).
Impact of phloem transport and dietary circumstances on seed storage protein accumulation
Synthesis of SSPs is dependent upon sugars and amino acids translocated from supply leaves through the phloem. The buildup of SSPs is subsequently influenced by plant metabolic standing and dietary circumstances. Expression of a soybean SSP was affected by sucrose provide (Bray and Beachy, 1985) and a potato tuber storage protein, patatin, has additionally been proven to answer sucrose (Martin et al., 1997). Equally, sugar transport to seed is more likely to have an effect on expression of SSP gene expression in Arabidopsis, though no direct proof supporting this speculation has been reported. Current evaluation on sugar-induced sign transduction revealed an involvement of hexokinase, SNF1 kinase, and regulatory PRL1 protein (Smeekens, 2000, for overview).
As described above, the Arabidopsis abi3 mutation impacts SSP gene expression. Along with this direct impact on SSPs, vegetation carrying the abi3 mutation additionally partitioned extra assets into seed growth than the wild kind. These further assets have been out there because of delayed senescence of the cauline leaves within the mutant. The change in distribution of photosynthate was confirmed by tracer evaluation (Robinson and Hill, 1999).
Lipid metabolism can be essential for expression of a category of SSPs. Transcription of the gene for oleosin throughout seed growth was delayed and diminished in an Arabidopsis mutant carrying a lesion affecting diacylglycerol acyltransferase, and not using a discount within the degree of oleosin protein in seeds, suggesting the presence of a coordinated regulation of lipid biosynthesis-regulated gene expression (Zou et al., 1996). Expression of oleosin was additionally affected each by way of timing and ranges by different mutations affecting seed growth resembling fus3 (Kirik et al., 1996).
Amino acids play one other essential function within the accumulation of SSPs. The broad specificity H+-amino acid co-transporter AAP1 is expressed within the endosperm and the cotyledons, whereas the same transporter AAP2 is expressed in vascular strands of siliques and in funiculi. These transporters are implicated to be concerned in long-distance transport of amino acids to seeds (Hirner et al., 1998). As well as, transgenic Arabidopsis vegetation overexpressing sense or antisense genes for a peptide transport gene, AtPTR2-B have been described. All 4 antisense traces and one sense line exhibited vital phenotypic adjustments, together with late flowering and arrested seed growth, suggesting a task of AtPTR2-B in nitrogen diet supply to seeds (Tune et al., 1997).
Sulfur diet has additionally been proven to have an effect on SSP accumulation. Typically, SSPs with a excessive content material of sulfur-containing amino acids accumulate to a decrease degree below circumstances of restricted sulfur provide and, conversely, accumulation of SSPs with low contents of sulfur-containing amino acids are elevated (Fujiwara et al., 1992). The SSPs of Arabidopsis have comparatively excessive contents of sulfur-containing amino acids and the buildup of 2S and 12S proteins below sulfur deficiency appear to be considerably downregulated (Naito et al., 1994a). The b conglycinin SSP of soybean is a properly studied instance of SSPs responding to dietary circumstances. Underneath a restricted provide of sulfur, accumulation of the b subunit of b-conglycinin is elevated and that of glycinin, one other main SSP, is downregulated (Gayler and Sykes, 1985). Utility of Met, then again, ends in a rise within the accumulation of glycinin and reduces or suppresses accumulation of the b subunit (Holowach et al., 1984). Transgenic Arabidopsis have been used successfully to look at the mechanisms of sulfur-regulated expression of soybean SSPs. Just like that in soybean, expression of the b subunit gene of b-conglycinin is upregulated by sulfur deficiency (Hirai et al., 1995) and downregulated by Met utility (Hirai et al., 1994) or mto1 mutations (Naito et al., 1994b; Naito et al., 1995, for overview) inflicting an overaccumulation of soluble Met (Inaba et al., 1994; Chiba et al., 1999) in transgenic Arabidopsis. The gene promoter of the b subunit of b-conglycinin has been proven to be chargeable for this regulation. Regulation of the b subunit promoter by the mto1 mutation is maternally managed, i.e., expression of the b-subunit promoter is set by the genotype of the feminine guardian whereas the genotype of the male guardian has little or no impact on the expression (Naito et al., 1994b). These findings recommend that Met, or certainly one of its metabolites, transported from the maternal tissues impacts SSP gene expression.
Expression of the b-subunit promoter in transgenic Arabidopsis was not solely regulated in line with sulfur diet, but in addition in line with nitrogen diet (Kim et al., 1999). Sulfur and nitrogen assimilation pathways merge on the step of cysteine biosynthesis and a precursor for cysteine biosynthesis, O-acetyl-L-serine (OAS), has been proven to be gathered relying on sulfur and nitrogen availability (Kim et al., 1999). Sulfur deficiency ends in a rise within the accumulation of OAS, whereas nitrogen deficiency decreases OAS accumulation. This sample was just like that for the b-subunit gene regulation (Kim et al., 1999). Furthermore, exogenous utility of OAS within the presence of excessive sulfate within the media induced accumulation of the b subunit in cultured soybean cotyledons (Kim et al., 1999). These findings recommend that OAS acts as a key metabolite controlling the expression of SSP genes in response to sulfur and nitrogen availability within the setting. It’s also intriguing that OAS contents have been elevated by ABA utility in immature soybean cotyledons (Kim et al., 1997). ABA utility to immature soybean cotyledon induces the same response to that of sulfur deficiency, i.e., a rise in b subunit accumulation and a lower in glycinin accumulation (Determine 4), suggesting a doable involvement of OAS within the ABA response.
“where is protein stored”