**2.1. Development of seed quality biomarkers**

Rajjou et al*.* [20] argued that characterizing biomarkers of seed vigor as a component of breeding programs is an important strategy for producing seeds of the highest possible quality, particularly under the environmental stresses occasioned by climate change and increasing world population. Thus developing bio-markers for seed aging and vigor loss traits are the first notable achievements of various seed vigor *-omics* projects. **Table 1** summarizes key *-omics*-based biomarkers of seed aging and/or vigor that have gained significant attention in the seed research community.

Advances in *-omics* sciences have made the acquisition of seed aging signals feasible so that a number of sensitive and effective biomarkers has been developed to identify aging signals and evaluate aging status [26]. Fu et al*.* [27] summarized *-omics* based biomarkers for seed aging signals of 17 species and classified them into six categories namely: molecular, biochemical, physiological, metabolic, mitochondrial and morphological signals (**Table 1**). Catusse et al*.* [15] used comparative proteomics to identify 18 proteins during seed priming, germination and aging. Bentsink et al. [28] showed that the *DOG1* gene that controls seed dormancy in Arabidopsis is also a biomarker for seed longevity since the mutations within the *DOG1* gene, specifically were associated with a seed longevity phenotype. Prieto-Dapena et al*.* [29] found that transgenic Arabidopsis seeds that over-accumulate a heat stress transcription factor exhibit a heat shock protein (*HSP*) biomarker for enhanced longevity. Whereas Devaiah et al*.* [24] reported a high level of a membrane lipid-hydrolyzing phospholipase-D (*PLDα1*) as a biomarker for reduced seed longevity. Kranner et al*.* [30] proposed the concept of half-cell reduction potential (*E*GSSG/2GSH) of glutathione (*GSH*) an antioxidant that scavenge ROS which increases to more oxidizing values during viability loss as biomarker signaling cascades that trigger cell death. Nagel et al*.* [5] engaged the *GSH* redox method as a biomarker for seed aging in barley. The activity of the protein l-isoaspartyl methyltransferase (*PIMT*), an enzyme repairing abnormal l-isoaspartyl residues in aging proteins of Arabidopsis is increasingly becoming a common biomarker in *-omics* both seed deterioration (aging) and seed invigoration (priming) studies in a number of crop plants [13, 14, 16, 22, 31] (**Table 1**).

Reports on seed priming-based *-omics* experiments have also been sources of seed vigor biomarkers. Chen et al*.* [32] reported protein profiling of spinach (*Spinacia oleracea* cv. Bloomsdale) seeds during priming with −0.6 MPa PEG at 15°C. The results showed two groups of proteins: Type I *i.e.* 37 and 35-kDa proteins which are major proteins in unprimed seeds gradually


**Table 1.** Biomarkers of that have gained attention in *-omics'* dissection of seed aging/vigor.

chapter, I will be discussing some of the major advances made with specific *-omics* technologies towards dissecting the complex seed vigor traits in various crop plant species. I will also focus on the direction to which the current advances in seed vigor *-omics* might be pointing the seed industry in the near future. In discussing all the sections, I will pay attention to seed vigor reports from the experimental perspectives of seed deterioration (aging) and seed

Rajjou et al*.* [20] argued that characterizing biomarkers of seed vigor as a component of breeding programs is an important strategy for producing seeds of the highest possible quality, particularly under the environmental stresses occasioned by climate change and increasing world population. Thus developing bio-markers for seed aging and vigor loss traits are the first notable achievements of various seed vigor *-omics* projects. **Table 1** summarizes key *-omics*-based biomarkers of seed aging and/or vigor that have gained significant attention in

Advances in *-omics* sciences have made the acquisition of seed aging signals feasible so that a number of sensitive and effective biomarkers has been developed to identify aging signals and evaluate aging status [26]. Fu et al*.* [27] summarized *-omics* based biomarkers for seed aging signals of 17 species and classified them into six categories namely: molecular, biochemical, physiological, metabolic, mitochondrial and morphological signals (**Table 1**). Catusse et al*.* [15] used comparative proteomics to identify 18 proteins during seed priming, germination and aging. Bentsink et al. [28] showed that the *DOG1* gene that controls seed dormancy in Arabidopsis is also a biomarker for seed longevity since the mutations within the *DOG1* gene, specifically were associated with a seed longevity phenotype. Prieto-Dapena et al*.* [29] found that transgenic Arabidopsis seeds that over-accumulate a heat stress transcription factor exhibit a heat shock protein (*HSP*) biomarker for enhanced longevity. Whereas Devaiah et al*.* [24] reported a high level of a membrane lipid-hydrolyzing phospholipase-D (*PLDα1*) as a biomarker for reduced seed longevity. Kranner et al*.* [30] proposed the concept of half-cell reduction potential (*E*GSSG/2GSH) of glutathione (*GSH*) an antioxidant that scavenge ROS which increases to more oxidizing values during viability loss as biomarker signaling cascades that trigger cell death. Nagel et al*.* [5] engaged the *GSH* redox method as a biomarker for seed aging in barley. The activity of the protein l-isoaspartyl methyltransferase (*PIMT*), an enzyme repairing abnormal l-isoaspartyl residues in aging proteins of Arabidopsis is increasingly becoming a common biomarker in *-omics* both seed deterioration (aging) and seed invigora-

tion (priming) studies in a number of crop plants [13, 14, 16, 22, 31] (**Table 1**).

Reports on seed priming-based *-omics* experiments have also been sources of seed vigor biomarkers. Chen et al*.* [32] reported protein profiling of spinach (*Spinacia oleracea* cv. Bloomsdale) seeds during priming with −0.6 MPa PEG at 15°C. The results showed two groups of proteins: Type I *i.e.* 37 and 35-kDa proteins which are major proteins in unprimed seeds gradually

invigoration (priming).

266 Advances in Seed Biology

the seed research community.

**2.** *-Omics* **technologies for seed aging/priming**

**2.1. Development of seed quality biomarkers**

depleting as priming progresses, and Type II such as ∼20 kDa doublets proteins having an accumulation patterns opposite to Type I during priming. The depletion and/or accumulation of Type I and Type II proteins during germination constitute biomarkers for seed vigor behaviors for the species. The type I proteins seed vigor biomarkers were seed maturation proteins, such as late embryogenesis abundant (LEA) or dormancy related proteins (e.g. short-chain dehydrogenase). The depletion of these proteins during germination and priming was also reported to mark seed vigor genes at transcription and translation levels in *B. oleracea* [12].

Chen et al*.* [33] identified a set of transcriptomic biomarkers for seed germination and vigor from quantitative real-time polymerase chain reaction (qRT-PCR) gene expression studies during germination of maize and spinach seeds. Since seed germination involves seeds transiting from dry and physiologically inactive state to hydrated and active state, the expression of house-keeping reference genes (HKG) may alter during the transition. From the study, the HKGs identified as valid reference genes and hence seed vigor biomarkers were *Actdf*, *UBQ*, *βtub*, *18S*, *Act*, and *GAPDH*. The HKG *18S* notably maintained stability through the transition state and was stable for both maize and spinach.

As the biomarker capabilities of *PIMT* genes has been reported for seed aging studies, likewise some seed priming studies have reported PIMT as biomarkers for seed vigor [16, 34]. The potential importance of *PIMT* as one key candidate seed vigor biomarker will be discussed later in this chapter.

#### **2.2. -***Omics* **of regulatory mechanisms for seed aging/priming**

Studies from the '70s and '80s identified the roles of regulatory hormones like ABA and GAs in seed germination control through mutations in Arabidopsis [35, 36]. With the advent of *-omics* methodologies, a significant in-depth understanding of regulatory mechanisms signaling seed deterioration and invigoration has been gained and applies to crops [37, 34]. Changes in specific sequences of highly polymorphic genetic markers in aging rice [38], tomatoes [39] and wheat seeds [40] provide hints of molecular hints on genetic influences behind seed aging.

Scanning through recent molecular studies on seed deterioration or invigoration, the mechanisms regulating seed vigor can be summarized into three systems: repair, protection, and detoxification systems [3, 23, 34, 41−44]. Research advances in manipulation of the cellular repair system for seed invigoration has been more pronounced, often intertwining with detoxification system research. The most apparently forward-looking cellular repair studies came out of the search for mechanisms underpinning the extra-ordinary longevity of sacred lotus (*Nelumbo nucifera*) seeds, which was found to be due to the repair activities of abnormal l-isoaspartyl residues accumulated in proteins during seed aging by *PIMT* in Arabidopsis [31]. PIMT combats protein mis-folding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms thus repairing an enzyme system which likely works with other anti-aging pathways to eliminate deleterious protein products and enable successful seedling establishment in the phenotypes [22, 31]. Studies of the role of *PIMT* in seed invigoration and longevity enhancement has extended to other crops, most of them reporting similar results. An immuno-localization study on rice concluded that the distinct *OsPIMT* isoform expression in embryo and aleurone layers of transgenic rice revealed its role in the restriction of deleterious isoAsp and age-induced ROS accumulation to improve seed vigor and longevity [14]. *PIMT* and *PIMT2* contains two genes (*At3g48330* and *At5g50240*) encoding protein-l-isoaspartate methyltransferase located on chromosome 5 and produces two proteins differing by three amino acids reported in Arabidopsis [22, 31], chickpea [13] and rice [16, 14]. The activities of two important *PIMT* coding genes (*At3g48330* and *At5g50240*) are gradually forming the bedrock for seed aging *-omics* and genetic engineering of seed vigor in many crop species [13, 22, 34].

Several *-omics* studies have generated information on the protective regulatory mechanism of seed aging/vigor. Protein and enzymes regulatory systems that are active in structural, membrane and genomic integrity were engaged in most of the studies that were attempting to dissect the protective system for seed invigoration [24, 29, 45]. Transcriptomics analysis of *de-novo* protein synthesis during priming-enhanced seed germination had shown the expression of aquaporins (AQPs) in abundance [46, 47]. AQPs are plasma membrane proteins known to regulate water transport, since they are mostly expressed in hydrated seeds. AQPs are either plasma membrane intrinsic proteins (−PIPs) or tonoplast intrinsic proteins (−TIPs) serving as water channels in membranes that control cell-to-cell water movement, plant cell expansion and organ development. The expression of four spinach (*S. oleracea*) AQP coding genes (SoPIP1;1, SoPIP1;2, SoPIP2;1, and SoδTIP) during osmopriming and germination under chilling drought and optimal conditions were investigated by [46]. The up-regulation of the four genes within 2–4 days of priming (phase II-imbibition) suggests that these proteins are essential for radicle protrusion and subsequent progress of seed germination vigor. The expression of vacuolar aquaporin genes increases a thousand times after the initiation of cell elongation in both orthodox and recalcitrant seeds [47]. During priming of *Beta vulgaris* L. (sugarbeet) seeds, Catusse et al*.* [15] used comparative proteomics to reveal 18 proteins exhibiting up-regulation during priming and down-regulation during aging and up-regulation again upon priming of the aged seeds. In the study, six translation initiation factors were found among the proteins exhibiting the highest levels of up-regulation upon priming the aged seeds, highlighting the roles of stored mRNAs and *de-novo* synthesized mRNAs in seed vigor protection regulatory mechanism. Proteomic analysis of sugarbeet seeds led to the identification of 758 proteins whose metabolic status in seed longevity protection can be inferred and reconstructed in further details [15]. Dinkova et al. [23] streamlined the translational control of seed germination in maize using the ratio of two cap binding proteins (*eIF(iso)4E* to *eIF4E*) in the corresponding eIF4F complex, *eIF(iso)4E* being more abundant in dry seeds and both cap-binding proteins being present at similar levels following 24-hour seed imbibition. Furthermore, Prieto-Dapena et al*.* [29] found that over-accumulation of heat stress transcription factor (HSPs) enhanced seed longevity in transgenic Arabidopsis seeds. Regente et al. [41] reported regulation of phospholipid accumulation in extracellular fluids of sunflower during priming and seed germination. Devaiah et al. [24] reported that the ablation of the gene for a membrane lipid-hydrolyzing phospholipase D (PLDα1) in Arabidopsis enhanced seed germination and oil stability after storage or exposure of seeds to adverse conditions. The *PLDα1*-deficient seeds exhibited a smaller loss of unsaturated fatty acids and lower accumulation of lipid peroxides than did wild-type seeds. However, *PLDα1*-knockdown seeds were more tolerant of aging than were *PLDα1*-knockout seeds. The results demonstrate the *PLDα1* plays an important role in seed deterioration and aging in Arabidopsis. A high level of *PLDα1* is detrimental to seed quality, and attenuation of *PLDα1* expression has the potential to improve oil stability, seed quality and seed longevity.

**2.2. -***Omics* **of regulatory mechanisms for seed aging/priming**

268 Advances in Seed Biology

Studies from the '70s and '80s identified the roles of regulatory hormones like ABA and GAs in seed germination control through mutations in Arabidopsis [35, 36]. With the advent of *-omics* methodologies, a significant in-depth understanding of regulatory mechanisms signaling seed deterioration and invigoration has been gained and applies to crops [37, 34]. Changes in specific sequences of highly polymorphic genetic markers in aging rice [38], tomatoes [39] and wheat seeds [40] provide hints of molecular hints on genetic influences behind seed aging. Scanning through recent molecular studies on seed deterioration or invigoration, the mechanisms regulating seed vigor can be summarized into three systems: repair, protection, and detoxification systems [3, 23, 34, 41−44]. Research advances in manipulation of the cellular repair system for seed invigoration has been more pronounced, often intertwining with detoxification system research. The most apparently forward-looking cellular repair studies came out of the search for mechanisms underpinning the extra-ordinary longevity of sacred lotus (*Nelumbo nucifera*) seeds, which was found to be due to the repair activities of abnormal l-isoaspartyl residues accumulated in proteins during seed aging by *PIMT* in Arabidopsis [31]. PIMT combats protein mis-folding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms thus repairing an enzyme system which likely works with other anti-aging pathways to eliminate deleterious protein products and enable successful seedling establishment in the phenotypes [22, 31]. Studies of the role of *PIMT* in seed invigoration and longevity enhancement has extended to other crops, most of them reporting similar results. An immuno-localization study on rice concluded that the distinct *OsPIMT* isoform expression in embryo and aleurone layers of transgenic rice revealed its role in the restriction of deleterious isoAsp and age-induced ROS accumulation to improve seed vigor and longevity [14]. *PIMT* and *PIMT2* contains two genes (*At3g48330* and *At5g50240*) encoding protein-l-isoaspartate methyltransferase located on chromosome 5 and produces two proteins differing by three amino acids reported in Arabidopsis [22, 31], chickpea [13] and rice [16, 14]. The activities of two important *PIMT* coding genes (*At3g48330* and *At5g50240*) are gradually forming the bedrock for seed

aging *-omics* and genetic engineering of seed vigor in many crop species [13, 22, 34].

Several *-omics* studies have generated information on the protective regulatory mechanism of seed aging/vigor. Protein and enzymes regulatory systems that are active in structural, membrane and genomic integrity were engaged in most of the studies that were attempting to dissect the protective system for seed invigoration [24, 29, 45]. Transcriptomics analysis of *de-novo* protein synthesis during priming-enhanced seed germination had shown the expression of aquaporins (AQPs) in abundance [46, 47]. AQPs are plasma membrane proteins known to regulate water transport, since they are mostly expressed in hydrated seeds. AQPs are either plasma membrane intrinsic proteins (−PIPs) or tonoplast intrinsic proteins (−TIPs) serving as water channels in membranes that control cell-to-cell water movement, plant cell expansion and organ development. The expression of four spinach (*S. oleracea*) AQP coding genes (SoPIP1;1, SoPIP1;2, SoPIP2;1, and SoδTIP) during osmopriming and germination under chilling drought and optimal conditions were investigated by [46]. The up-regulation of the four genes within 2–4 days of priming (phase II-imbibition) suggests that these proteins Cellular detoxification mechanisms have been widely viewed as an important mechanism for seed invigoration. Several gene activities have been identified that controls these mechanisms [48, 49]. Detoxification genes/proteins that scavenge ROS are the most investigated system for mechanism for seed vigor enhancement. For example, Nagel et al*.* [5] linked seed aging to genetic backgrounds that regulate the production of ROS-scavenging antioxidants which are known to detoxify aging cells to enhance vigor in a similar fashion to cellular repair mechanisms. Antioxidants such as glutathione (GSH), tocochromanols and ascorbic acid scavenge ROS [42]. Decreases in the antioxidant capacity of GHS under continuous accelerated aging stress increase ROS, shifting the antioxidant redox state towards more oxidizing conditions. In agreement with this concept, the glutathione half-cell reduction potential (*EGSSG/2GSH*) increases to oxidizing values during viability loss, which is assumed to initiate further signaling cascades that trigger cell death [30]. The accumulation of oxidative damage in seeds was correlated with seed vigor loss [26]. At the molecular level, the process of carbonylation, in other words, increased protein oxidation often induces loss of functional properties of target seed proteins or enzymes thus increasing their susceptibility to proteolysis. Since the presence of ROS attacks proteins by oxidizing them, the important role of antioxidant systems through detoxification and protection of upstream mechanisms to maintain seed vigor is underscored.

#### **2.3. Mapping the genes controlling seed aging/vigor**

The use of molecular markers in modern plant breeding to increase selection efficiency through mapping genes to specific traits of interest was made possible by *-omics* precision tools. For many simply inherited traits of economic importance, fine-mapping and tagging with closely linked or gene-specific markers is straightforward simple. However, seedling vigor in crop plants is a complex quantitative trait under the control of large genotype and environment (GxE) effects. Hence, the advent of genomics tools for mapping and analyzing quantitative trait loci (QTL) is a major breakthrough for breeding seed vigor traits and gene identification for further experimentation. From many seed deterioration experiments, QTLs of seed longevity traits like LD50 in Arabidopsis seeds [21], germination of aged wheat seeds [48, 49] and half-life (P50) of aging barley seeds [50] have been mapped and linked to various genes. For the germination vigor of seeds, several seed priming experiments have found QTLs for germination of maize seeds [51] and QTLs for 30 vigor traits of rice seeds [52] to mention a few. These studies also provided useful information on chromosome regions and putative genes controlling various seed vigor traits in different crops.

QTL work on seed vigor began with the pioneering work of Clerkx et al*.* [21] on the model plant Arabidopsis, where QTL mapping was used to identify the loci controlling various aspects of seed longevity during storage and germination. Genotyping a recombinant inbred line population with 65 PCR-based markers and seed LD50 of phenotypic marker erecta, they identified three QTLs affecting seed longevity after controlled deterioration on chromosomes 1, 3, and 4 for Arabidopsis. Nagel et al*.* [50] also reported large QTL effects associated with seed half-life (P50) on chromosomes 5 and 7 in a doubled haploid mapping population of barley. Han et al*.* [51] found 65 QTLs in two maize populations mapped using single-nucleotide polymorphism (SNP) markers to four seed vigor traits under four germination treatment conditions. Integrating the QTLs into 18 meta-QTLs (mQTLs), 23 candidate genes associating with seed vigor phenotype coincides with 13 mQTLs controlling protein metabolism and the glycolytic pathway. They reported four seed vigor hotspots on chromosome regions for mQTL2, mQTL3-2, mQTL3-4, and mQTL5-2 with large QTL effects under various germination environments. There are a number of recent QTL studies on seed vigor of rice [52, 53]. Singh et al*.* [52] reported seed germination capacity of primed rice seeds derived from 253 BC3 F4 lines of crosses between Swarna and Moroberekan, phenotyped for early vigor and genotyped with 194 SNP markers. They identified six seed vigor genomic regions on chromosomes 3, 4, 5, and 6 [52]. Two of the QTL regions namely chr3 (*id3001701*-*id300833*) and chr5 (*wd5002636-id5001470*) were identified and tagged QTL hotspots because they were expressed consistently in field and glasshouse conditions. In the chr3 hotspot, most of QTLs identified for early vigor-related traits were *qEV3.1, qEUE3.1, qSHL3.1, qSL3.1, qSFW3.1, qTFW3.1, qRDW3.1* associated with early vigor, early uniform emergence, shoot length, stem length, shoot fresh weight, total fresh weight and root dry weight respectively. The QTL hotspot on chr5 includes almost similar seed vigor traits as the first hotspot except total fresh weight and root dry weight but includes seed dry weight (*qSDW5.1*) and total dry weight (*qTDW5.1*).

From these QTL regions identified in the brief review above, putative candidate genes associated with many seed vigor traits in the hotspot QTL regions have been published for crops like wheat [35], maize [38] and rice [40]. Besides, Carrera et al*.* [54] used gene expression profiling *-omics* method to produce a list of candidate genes that signify seed germination which was used to produce TAGGIT, a spreadsheet based seed specific gene ontology that describes the seed germination signature. Other seed specific genomic resources for seed vigor are: PageMan/MapMan package which visualizes transcriptome changes in *Arabidopsis* [55] seeds during germination, and SeedNet which describes transcriptional interactions for seed vigor regulation [56].
