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Seed development in Arabidopsis thaliana undergoes an initial phase of endosperm proliferation followed by a second phase in which the embryo grows at the expense of the endosperm. As mature seed size is largely attained during the initial phase, seed size is coordinately determined by the growth of the maternal ovule, endosperm, and embryo. Here, we identify SHORT HYPOCOTYL UNDER BLUE1 (SHB1) as a positive regulator of Arabidopsis seed development that affects both cell size and cell number. shb1-D, a gain-of-function overexpression allele, increases seed size, and shb1, a loss-of-function allele, reduces seed size. SHB1 is transmitted zygotically. The increase in shb1-D seed size is associated with endosperm cellurization, chalazal endosperm enlargement, and embryo development. SHB1 is required for the proper expression of two other genes that affect endosperm development, MINISEED3 (MINI3) and HAIKU2 (IKU2), a WRKY transcription factor gene and a leucine-rich repeat receptor kinase gene. SHB1 associates with both MINI3 and IKU2 promoters in vivo. SHB1 may act with other proteins that bind to MINI3 and IKU2 promoters to promote a large seed cavity and endosperm growth in the early phase of seed development. In the second phase, SHB1 enhances embryo cell proliferation and expansion through a yet unknown IKU2-independent pathway. INTRODUCTIONIn angiosperms, double fertilization leads to the formation of a diploid embryo and a triploid endosperm, as the endosperm arises from the central cell that contains two identical haploid genomes. The endosperm constitutes the major volume of the mature seed in monocots and some dicots. In Arabidopsis thaliana and many other dicots, seed development is marked by two distinct phases (Sundaresan, 2005). In an initial phase, rapid growth and proliferation of the endosperm results in a large increase in size (Boisnard-Lorig et al., 2001). Then, embryo growth takes place at the expense of endosperm during the second phase. At maturity, the seed contains only a single layer of endosperm cells, and the maternal integument ultimately becomes the seed coat (Scott et al., 1998;Garcia et al., 2003). Seed coat formation and endosperm growth precede embryo growth in Arabidopsis, and the seed reaches almost its final size before the enlargement of the embryo. Therefore, seed size is determined by the coordinated growth of the diploid embryo, the triploid endosperm, and the diploid maternal ovule.Both maternal and nonmaternal factors are involved in seed size regulation (Garcia et al., 2005). In Arabidopsis, an increased dosage of the paternal genome in the endosperm increases seed size, whereas an increased dosage of the maternal genome reduces seed size, with delayed cellularization of the peripheral endosperm and hypertrophy of the chalazal endosperm and associated nodules (Scott et al., 1998). Mutations in DNA METHYLTRANSFERASE1 (MET1) and DECREASE IN DNA METHYLATION1 (DDM1) dramatically reduce DNA methylation and cause parent-of-origin effects on F1 seed size (Xiao et al., 2006). Poll...

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Photomorphogenesis is regulated by red/far-red light-absorbing phytochromes and blue/UV-A light-absorbing cryptochromes. We isolated an Arabidopsis thaliana blue light mutant, short hypocotyl under blue1 (shb1), a knockout allele. However, shb1-D, a dominant allele, exhibited a long-hypocotyl phenotype under red, far-red, and blue light. The phenotype conferred by shb1-D was caused by overaccumulation of SHB1 transcript and recapitulated by overexpression of SHB1 in Arabidopsis. Therefore, SHB1 acts in cryptochrome signaling but overexpression may expand its signaling activity to red and far-red light. Consistent with this, overexpression of SHB1 enhanced the expression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) under red light. PIF4 appears to specifically mediate SHB1 regulation of hypocotyl elongation and CHLOROPHYLL a/b BINDING PROTEIN3 or CHALCONE SYNTHASE expression under red light. Overexpression of SHB1 also promoted proteasome-mediated degradation of phytochrome A and hypocotyl elongation under far-red light. Under blue light, shb1 suppressed LONG HYPOCOTYL IN FAR-RED LIGHT1 (HFR1) expression and showed several deetiolation phenotypes similar to hfr1-201. However, the hypocotyl and cotyledon-opening phenotypes of shb1 were opposite to those of hfr1-201, and HFR1 acts downstream of SHB1. SHB1 encodes a nuclear and cytosolic protein that has motifs homologous with SYG1 protein family members. Therefore, our studies reveal a signaling step in regulating cryptochromeand possibly phytochrome-mediated light responses.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_-Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Acellular biological tissues, including bovine pericardia (BP), have been proposed as natural biomaterials for tissue engineering. However, small pore size, low porosity and lack of extra cellular matrix (ECM) after native cell extraction directly restrict the seed cell adhesion, migration and proliferation and which is a vital problem for ABP's application in the tissue engineered heart valve (TEHV). In the present study, we treated acellular BP with acetic acid, which increased the scaffold pore size and porosity and conjugated RGD polypeptides to ABP scaffolds. After 10 days of culture in vitro, the human mesenchymal stem cells (hMSCs) attached the best and proliferated the fastest on RGD-modified acellular scaffolds, and the cell has grown deep into the scaffold. In contrast, a low density of cells attached to the unmodified scaffolds, with few infiltrating into the acellular tissues. These findings support the potential use of modified acellular BP as a scaffold for tissue engineered heart valves.

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Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement.