**3. Genetic regulation during seed filling**

Filling occurs when embryonic development is complete in the seed. Seed filling is under genetic control and is tied to changes in storage reserves. Seed filling is regulated by a network of signals mediated by various hormones [54, 55]. Basic research and information on differentiation, growth, and signal transduction related to seed filling and development is derived from studies in *Arabidopsis thaliana*, and there are sufficient reports to suggest that the major mechanisms regulating filling and development are similar in all plant seeds [55–57]. Recent developments in molecular genetics and genomics have led to a better understanding of the processes that occur during seed filling and development and offer opportunities to control and modify seed quality as well [58]. In addition, understanding the genetic factors that influence seed development could help breeders manipulate filling rate and duration to obtain higher yielding varieties.

Plotting phenotypic values on the growth curve throughout the duration of seed filling and analyzing the results using quantitative genetic approaches is a good strategy for exploring a time-dependent trait [59] to understand seed filling. Gene expression regulating storage reserves during seed filling is interrelated [54, 55, 60]

and many known genes are expressed in the endosperm of flowering plants (**Table 1**). In *Arabidopsis*, four major regulators (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], LEAFY COTYLEDON1 [LEC1], and LEC2) control many aspects of seed development, such as the accumulation of storage molecules, cotyledon identity, and the transition to desiccation tolerance and dormancy [66]. The ABI3, FUS3, LEC1, and LEC2 network of regulators has the common phenotypic effect of reduced expression of seed storage proteins. In addition, the main role of the LEC2 regulatory network is to up-regulate *FUS3* and *ABI3.* In *Arabidopsis*, several genes have been identified, such as the *FERTILIZATION INDEPENDENT SEED* (*FIS*) genes *MEDEA* (*MEA*) [67, 68], *FERTILIZATION INDEPENDENT ENDOSPERM* (*FIE*) [69], *FIS2* and *MULTI-COPY OF IRA1* (*MSI1*) [70, 71], *MEA* homologs *CURLY LEAF* (*CLF*) or *SWINGER* (*SWN*) [72], and *MATERNALLY EXPRESSED PAB C-TERMINAL* (*MPC*) [73] and *FLOWERING WAGENINGEN* (*FWA*). These genes are all involved in seed filling and early seed development in plants [74, 75].

DNA methylation is also one of the first recognized epigenetic modifications that affect gene expression by determining chromatin structure and compartmentalization of DNA during seed filling [55, 76]. *METHYLTRANSFERASE1* (*MET1*) is the major methyltransferase gene in *Arabidopsis* [77] and DNA methylation by *MET1* is involved in epigenetic control of seed size [78]. Transcriptome dynamics during seed filling have been described in several crops. As observed in barley and wheat seeds, the transition from cellular differentiation to filling in rice seeds is associated with


#### **Table 1.**

*Some genes involved in seed filling regulation.*

#### *Seed Filling DOI: http://dx.doi.org/10.5772/intechopen.106843*

changes in gene expression patterns [79, 80]. Using microarray technology, more than 20.000 genes associated with seed filling have been identified in rice, many of them related to metabolic pathways of carbohydrates and fatty acids [81]. The results of cluster and correlation analysis of these genes revealed 269 genes associated with seed filling [81]. In alfalfa, cluster analysis identified 5165 genes involved in seed filling, and most of these genes were associated with metabolic pathways of proteins for seed storage [58, 82]. The major regulators of gene expression are miRNAs and their expression has been studied in some crops [83], including rice, wheat, and maize [83–85]. Members of the miR156 family are specifically expressed during seed filling in rice by targeting genes of the squamosal promoter-binding protein-like (*SPL*) family. One of these genes, *SPL16,* controls cell proliferation during seed filling, and increased expression of *SPL16* correlates positively with grain yield in rice [86]. miR397 enhances brassinosteroid signaling by down-regulating the *laccase* (*LAC*) gene, which increases grain yield in rice [87]. Also, the expression levels of miR156, miR164, miR166, miR167, and miR1861 suggest that they play regulatory roles in rice during seed development and filling [84].

The major storage reserves accumulated during seed filling are storage proteins, lipids (generally TAGs), and carbohydrates (generally starch). Regulatory networks controlling seed filling are repressed prior to germination to prevent the accumulation of reserves during the vegetative development. Therefore, studies of gene expression during seed filling in tissues at the vegetative developmental stage may provide insight into the regulatory mechanisms underlying seed filling. The transcription factor WRINKLED1 (WRI1) plays an important role in fatty acid accumulation during seed development in *A. thaliana* [88]. Genetic and molecular studies suggest that WRI1 is a target of *LEC2*, *ABI3, FUS3*, and *LEC2* to regulate oleosin expression and lipid accumulation [88]. In *Arabidopsis*, mutations in *LEC1, LEC2* and *FUS3* resulted in decreased accumulation of storage proteins and TAGs [89, 90]. Synthesis of fatty acids in lipid metabolism during seed filling occurs through stimulation of fatty acid synthase or acyl carrier protein genes [58].

Sugar molecules can act as signaling molecules that regulate genes expressed in photosynthesis and metabolism. High sugar content promotes starch biosynthesis, while it has a negative effect on photosynthesis. Low sugar content increases the expression of genes related to photosynthesis and promotes the transport of seed reserves, while it limits the metabolic processes of carbohydrates [91]. Sucrose content controls cell differentiation and filling processes in seeds by altering gene expression and enzyme activities [92]. In faba bean, pea, and barley seeds, sucrose initiates gene expression regulating seed storage reserves and triggers the transition from embryogenesis to seed filling [93, 94]. Sucrose induces gene expression of globulin and albumin proteins, and *LEC1*, *LEC2* and *FUS3* are important regulators of sucrose in *Arabidopsis* [95]. Sucrose is also imported and converted to starch in endosperm during seed filling [96]. In *Arabidopsis*, mutations in sucrose transporter gene (*AtSUC5*) delayed the conversion of sugars to lipids, and the *AtSUC5* gene is involved in seed filling [97]. In rice, *AGPS2/shrunken2* (starch synthesis gene) is upregulated during the period of increasing seed dry weight [98]. Two rice sucrose synthase (*SUS*) genes are expressed, one at the early stage of seed filling and the other during seed filling, but not in the endosperm [81]. The *GRMZM2G391936* and *GRMZM2G008263* genes are involved in starch and sucrose metabolism during seed filling in maize [59]. The gene *GRMZM2G008263* is the starch synthase gene responsible for the production of amylose and is found only in starch grains [59]. The *GRMZM2G391936* gene encodes the large subunit of ADP-glucose pyrophosphorylase (AGPase). Alteration

of AGPase activity can increase the yield of starchy plants [99–101]. *Trehalose-6 phosphate synthase1* (*tps1*) mutants demonstrated the importance of sugar signaling molecules during seed filling by down-regulating genes for starch-sucrose degradation and up-regulating genes for lipid mobilization to produce glucose [102]. Therefore, sugars as signaling molecules are important regulators during seed filling.

The amino acid content and the composition of the seed storage proteins influence the nutritional value of the seeds. Storage proteins are synthesized during seed filling and deposited in endosperm tissues. The rate of amino acid synthesis controls the rate and yield of storage protein synthesis. The phosphoenol pyruvate carboxylase (PEPC) enzyme is a critical factor in the biosynthesis of storage proteins in soybean, pea, faba bean, and wheat. Therefore, PEPC can be used to increase the protein content of seeds. Overexpression of PEPC in bean seeds results in up to 20% higher protein content per gram dry weight due to increased sugar/starch and free amino acid content [103], which led to the identification of an important marker for the transition from seed filling to the drying stage. Up-regulation of genes involved in amino acid metabolism (such as the amino-transferase gene) during seed filling in alfalfa results in increased amino acid synthesis, which is required for the production of seed storage proteins [58]. In maize, the expression level of marker genes for amino acid synthesis during seed filling has been studied [104]. One of these genes, *ZmAS1,* was expressed in both cobs and kernels, while others, *ZmAS2* and *ZmAS3,* were expressed in kernels. In alfalfa seeds, most of the storage reserves accumulate between 14 and 36 DAP in the embryo at the seed filling stage [105]. The *stress-associated protein 1* (*MtSAP1*) gene of alfalfa directly regulates the accumulation of seed storage proteins [106]. Phaseolin is the major seed storage protein in bean and the phaseolin (*phas*) gene is not expressed during the vegetative phase of plant development [107].

Genes responsible for the accumulation of storage proteins and lipids during seed filling are controlled by cis-acting elements in promoters. Well-characterized ciselements are the RY repeats (CATGCA), the ACGT box (CACGTG), and the AACA motifs, controlled by the B3, bZIP, and MYB domain transcription factors, respectively [55]. For example, silencing of the *phas* gene in vegetative tissues has been associated with the presence of TATA boxes in the *phas* promoter [108].

Abscisic acid (ABA) is a key hormone involved in the regulation of several processes of seed development, such as maturation and reserve accumulation [109]. In *Arabidopsis*, barley and bean seeds *CYP707A* genes regulate ABA degradation in the embryo and endosperm [110–112]. In addition, gibberellins (GA) and ABA are also involved in cell differentiation and grain filling processes [112, 113]. While the level of GA is suppressed during seed filling, the level of ABA increases. In *Arabidopsis*, the biosynthesis of GA is controlled by the expression of the *AtGA2ox6* gene, but its expression is controlled by ABA levels [114]. Auxin is also involved in the seed filling process and interacts with the transcription factors LEC2 and FUS3 [55, 115]. The transcription factor ABI3 is involved in auxin signaling [116]. Expression of *LEC2* activates auxin-related genes [117] and auxin activates the expression of *FUS3* [118].

Flavonoids such as proanthocyanidins and anthocyanins are accumulated in seeds during seed filling. In alfalfa, several genes such as *MtWD40-1* [119], *MtMYB5* and *MtMYB14* [120], *MtPAR* (a regulator of proanthocyanidin accumulation through its effect on *MtWD40-1*; [121]) have been identified to be involved in the proanthocyanidin biosynthetic pathway during seed filling. In addition, genes for flavonoid biosynthesis have been identified, including chalcone synthases (*Mtr.42237.1.S1\_at*), chalcone isomerases (*Mtr.40331.1.S1\_at*), and flavonol synthases (*Mtr.45897.1.S1\_at*) in alfalfa [58]. These enzymes cause the accumulation of isoflavones in the embryo

#### *Seed Filling DOI: http://dx.doi.org/10.5772/intechopen.106843*

[122], but may also be involved in the accumulation of proanthocyanidins in the seed coat and tannins in the bark tissues [123]. Other genes involved in the accumulation of proanthocyanidins have been identified, such as glycosyltransferase (*UGT72L1*) and the proanthocyanidin transporter *MATE1* [124], which is responsible for the synthesis and modification of proanthocyanidin precursors [125]. *MATE2* transports anthocyanin by diverting flavonoid precursors into the anthocyanin pathway [126]. In addition, glycosyltransferase (*UGT78G1*) is required for the modification and accumulation of anthocyanins [127]. All of these genes are involved in the control of anthocyanidin reductase, one of the major enzymes responsible for the production of proanthocyanidins.
