STENOFOLIARegulates Blade Outgrowth and Leaf Vascular Patterning inMedicago truncatulaandNicotiana sylvestris

Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes.plant organ size | seed size | forage quality | Medicago | soybean A s a key crop worldwide, soybean not only provides up to 69% of proteins and 30% of oils to the human diet (1) but also requires a low input of fertilizers due to its symbiotic nitrogen fixation ability (2), making it a highly valuable crop to secure global food supplies while contributing to sustainable agriculture. It is clear that the rapid increase of world population requires significant increases in crop production (3).Seed size is a major agronomic trait that has been selected in crop plants during their domestication (4-6). Due to whole genome duplication events that occurred ∼59 and ∼14 Mya (million years ago) (2), soybean has a complex genome structure. Thus, the isolation of key genes or quantitative trait loci (QTL) that control seed size and weight in soybean using conventional approaches can be a challenge and has not been reported to date. To overcome this challenge, we took a molecular genetics approach, using the legume plant Medicago truncatula as a genetic model. We identified BIG SEEDS1 (BS1) as a key gene that controls size of lateral organs, including seeds, seed pods, and leaves, in M. truncatula. Based on these results, we further identified two BS1 orthologs in soybean (Glycine max). Our objectives were to understand the role and regulatory mechanism of the BS1 gene in lateral organ size control in legume plants. Down-regulation of soybean BS1 genes using an artificial microRNA resulted in increased size of seeds, seed pods, and leaves, thus revealing a key and conserved role of BS1 in the control of organ size in legumes. Results and DiscussionIsolation and Characterization of M. truncatula big seeds1 Mutants.In a screen of the fast neutron bombardment (FNB)-induced deletion mutant collection of M. truncatula [cultivar (cv.) Jemalong A17] (7), a unique mutant with large seeds was isolated and named M. truncatula big seeds1-1 (mtbs1-1) (Fig. 1A). The mtbs1-1 mutant also exhibited larger seed pods and leaves than WT plants, suggesting a key role of BS1 in determining lateral organ size (SI Appendix, Figs. S1 and S2). Time...

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“…M. truncatula transcriptomic analysis was performed as previously described (32,33). See SI Appendix for details.…”

Section: Methodsmentioning

confidence: 99%

Increasing seed size and quality by manipulating BIG SEEDS1 in legume species

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The lamina and flower phenotypes of lam1 mutants can be complemented by Arabidopsis WUS when expressed under the control of the STF promoter, suggesting that a WUS-like function is required for cell proliferation in determinate lateral organs (15,17). We conducted the present study to determine the mechanism of STF/LAM1 function and establish the molecular basis of WUS-mediated complementation of mutant phenotypes in lateral organs.…”

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confidence: 99%

“…These mutants produce narrow leaf blades owing to defects in lateral cell proliferation affecting leaf width, whereas leaf length is virtually unaffected. In leaf primordia, STF/MAW/ WOX1 and PRS/WOX3 are expressed at the adaxial-abaxial boundary layer both in the middle mesophyll and at the leaf margin (14)(15)(16), indicating that this region may be important for blade outgrowth. Arabidopsis WOX1 and PRS are proposed to coordinate adaxial/abaxial patterning through interaction with adaxial and abaxial domain-specific polarity factors (16).…”

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confidence: 99%

“…NS1 and NS2 are homologs of the Arabidopsis gene PRS/WOX3 involved in the development of lateral sepals and stipules (11,13). Recent work has identified narrow leaf mutants in the WOX1 homologs maewest (maw) in Petunia x hybrida (14), stenofolia (stf) in Medicago truncatula (15), lam1 in Nicotiana sylvestris (15), and wox1/prs double mutant in Arabidopsis thaliana (14,16). These mutants produce narrow leaf blades owing to defects in lateral cell proliferation affecting leaf width, whereas leaf length is virtually unaffected.…”

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confidence: 99%

“…In M. truncatula and N. sylvestris, loss-of-function mutations in STF and LAM1, respectively, are sufficient to arrest lateral growth, leading to severe defects in lamina outgrowth, vein patterning, petal lobe expansion, and ovary fusion (15). The lamina and flower phenotypes of lam1 mutants can be complemented by Arabidopsis WUS when expressed under the control of the STF promoter, suggesting that a WUS-like function is required for cell proliferation in determinate lateral organs (15,17).…”

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confidence: 99%

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Evolutionarily conserved repressive activity of WOX proteins mediates leaf blade outgrowth and floral organ development in plants

Lin

Niu

McHale

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

137

169

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The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades-ancient, intermediate, and modern/WUS-based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUSbox of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box.T he WUSCHEL related homeobox (WOX) genes form a plantspecific family of the eukaryotic homeobox transcription factor superfamily (1, 2). In Arabidopsis, the WOX family consists of 15 members, including the founding member WUSCHEL (WUS) and WOX1-WOX14 (3), which are involved in the regulation of key developmental processes, including stem cell maintenance in shoot and root meristems, embryo apical-basal polarity patterning, and development of lateral organs (1, 4-7). Based on phylogenetic analysis and their distribution in the plant kingdom, WOX genes have been classified into three clades: modern/WUS (found in seed plants), intermediate (found in vascular plants including lycophytes), and ancient (found in vascular and nonvascular plants, including mosses and green algae) (1,8). WOX genes have been proposed to have a common mechanism of action, as demonstrated by complementation of the wox5 and pressed flower (prs/wox3) mutant phenotypes by WUS, as well as by the partial complementation of prs with WOX4 (5, 9, 10). However, the extent to which the functions of WOX family members are conserved remains unclear.The critical requirement of WOX genes for the development of lateral organs, including leaves and flowers, has become increasingly apparent from the identification of several mutant phenotypes in angiosperms. In maize, the narrowsheath1 and 2 (ns1/ns2) double...

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