Genome-Wide Identification and Expression Analysis of Growth-Regulating Factors in Eucommia ulmoides Oliver (Du-Zhong).

The roots, stems, leaves, and seeds of Eucommia ulmoides contain a large amount of trans-polyisoprene (also known as Eu-rubber), which is considered to be an important laticiferous plant with valuable industrial applications. Eu-rubber used in industry is mainly extracted from leaves. Therefore, it is of great significance to identify genes related to regulating the leaf size of E. ulmoides. Plant growth-regulating factors (GRFs) play important roles in regulating leaf size, and their functions are highly conserved across different plant species. However, there have been very limited reports on EuGRFs until now. In this study, eight canonical EuGRFs with both QLQ and WRC domains and two putative eul-miR396s were identified in the chromosome-level genome of E. ulmoides. It is found that, unlike AtGRFs, all EuGRFs contain the miR396s binding site in the terminal of WRC domains. These EuGRFs were distributed on six chromosomes in the genome of E. ulmoides. Collinearity analysis of the E. ulmoides genome revealed that EuGRF1 and EuGRF3 exhibit collinear relationships with EuGRF2, suggesting that those three genes may have emerged via gene replication events. The collinear relationship between EuGRFs, AtGRFs, and OsGRFs showed that EuGRF5 and EuGRF8 had no collinear members in Arabidopsis and rice. Almost all EuGRFs show a higher expression level in growing and developing tissues, and most EuGRF promoters process phytohormone-response and stress-induced cis-elements. Moreover, we found the expression of EuGRFs was significantly induced by gibberellins (GA3) in three hours, and the height of E. ulmoides seedlings was significantly increased one week after GA3 treatment. The findings in this study provide potential candidate genes for further research and lay the foundation for further exploring the molecular mechanism underlying E. ulmoides development in response to GA3.

GRAS transcription factor PINNATE-LIKE PENTAFOLIATA2 controls compound leaf morphogenesis in Medicago truncatula.

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation.

Control of compound leaf patterning by MULTI-PINNATE LEAF1 (MPL1) in chickpea.

Plant lateral organs are often elaborated through repetitive formation of developmental units, which progress robustly in predetermined patterns along their axes. Leaflets in compound leaves provide an example of such units that are generated sequentially along the longitudinal axis, in species-specific patterns. In this context, we explored the molecular mechanisms underlying an acropetal mode of leaflet initiation in chickpea pinnate compound leaf patterning. By analyzing naturally occurring mutants multi-pinnate leaf1 (mpl1) that develop higher-ordered pinnate leaves with more than forty leaflets, we show that MPL1 encoding a C2H2-zinc finger protein sculpts a morphogenetic gradient along the proximodistal axis of the early leaf primordium, thereby conferring the acropetal leaflet formation. This is achieved by defining the spatiotemporal expression pattern of CaLEAFY, a key regulator of leaflet initiation, and also perhaps by modulating the auxin signaling pathway. Our work provides novel molecular insights into the sequential progression of leaflet formation.

Genome-Wide Identification and Expression Analysis of the VILLIN Gene Family in Soybean.

The VILLIN (VLN) protein is an important regulator of the actin cytoskeleton, which orchestrates many developmental processes and participates in various biotic and abiotic responses in plants. Although the VLN gene family and their potential functions have been analyzed in several plants, knowledge of VLN genes in soybeans and legumes remains rather limited. In this study, a total of 35 VLNs were characterized from soybean and five related legumes. Combining with the VLN sequences from other nine land plants, we categorized the VLN gene family into three groups according to phylogenetic relationships. Further detailed analysis of the soybean VLNs indicated that the ten GmVLNs were distributed on 10 of the 20 chromosomes, and their gene structures and protein motifs showed high group specificities. The expression pattern analysis suggested that most GmVLNs are widely expressed in various tissues, but three members have a very high level in seeds. Moreover, we observed that the cis-elements enriched in the promoters of GmVLNs are mainly related to abiotic stresses, hormone signals, and developmental processes. The largest number of cis-elements were associated with light responses, and two GmVLNs, GmVLN5a, and GmVLN5b were significantly increased under the long light condition. This study not only provides some basic information about the VLN gene family but also provides a good reference for further characterizing the diverse functions of VLN genes in soybeans.

The Pea R2R3-MYB Gene Family and Its Role in Anthocyanin Biosynthesis in Flowers.

Pea (Pisum sativum L.) is one of the most important legume crops in the world, and it has attracted great attention for its high nutritive values. Recently, the crop breeding program has been focused on the crop metabolic engineering (i.e., color, flavor, nutrition) to improve the quality of crop. As a major group of transcription factors forming the ternary MYB-bHLH-WD repeat protein (MBW) complex to regulate the anthocyanin biosynthesis pathway, members of R2R3-MYB gene family have always been the focus of research targets to improve the valuable metabolic product of crops. Until now, few report about the R2R3-MYB gene family of pea has been released. In this study, we identified 119 R2R3-MYB genes in the assembled pea genome (Version 1a), of which 111 were distributed across 14 chromosomes. Combining with the 126 R2R3-MYB protein sequences of Arabidopsis, we categorized 245 R2R3-MYB proteins into 36 subgroups according to sequence similarity and phylogenetic relationships. There was no member from subgroup 12, 15 and 29 existing in pea genome, whereas three novel subgroups were found in pea and named as N1-N3. Further analyses of conserved domains and Motifs, gene structures, and chromosomal locations showed that the typical R2 and R3 domains were present across all R2R3-MYB proteins, and Motif 1, 2, and 3 were identified in most members. Most of them had no more than two introns. Additionally, 119 pea R2R3-MYB genes did not experience large-scale duplication events. Finally, we concluded that several candidate genes may be responsible for the spatiotemporal accumulation of anthocyanins in pea petals. PsMYB116 was predominantly expressed in the dorsal petals to presumably activate the anthocyanin biosynthesis pathway, while PsMYB37 and PsMYB32 may positively regulates the anthocyanin accumulation in the lateral petals. This study not only provides a good reference to further characterize the diverse functions of R2R3-MYB genes but also helps researchers to understand the color formation of pea flowers.

MtGSTF7, a TT19-like GST gene, is essential for accumulation of anthocyanins, but not proanthocyanins in Medicago truncatula.

Anthocyanins and proanthocyanins (PAs) are two end products of the flavonoid biosynthesis pathway. They are believed to be synthesized in the endoplasmic reticulum and then sequestered into the vacuole. In Arabidopsis thaliana, TRANSPARENT TESTA 19 (TT19) is necessary for both anthocyanin and PA accumulation. Here, we found that MtGSTF7, a homolog of AtTT19, is essential for anthocyanin accumulation but not required for PA accumulation in Medicago truncatula. MtGSTF7 was induced by the anthocyanin regulator LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1), and its tissue expression pattern correlated with anthocyanin deposition in M. truncatula. Tnt1-insertional mutants of MtGSTF7 lost anthocyanin accumulation in vegetative organs, and introducing a genomic fragment of MtGSTF7 could complement the mutant phenotypes. Additionally, the accumulation of anthocyanins induced by LAP1 was significantly reduced in mtgstf7 mutants. Yeast-one-hybridization and dual-luciferase reporter assays revealed that LAP1 could bind to the MtGSTF7 promoter to activate its expression. Ectopic expression of MtGSTF7 in tt19 mutants could rescue their anthocyanin deficiency, but not their PA defect. Furthermore, PA accumulation was not affected in the mtgstf7 mutants. Taken together, our results show that the mechanism of anthocyanin and PA accumulation in M. truncatula is different from that in A. thaliana, and provide a new target gene for engineering anthocyanins in plants.

The F-box protein MIO1/SLB1 regulates organ size and leaf movement in Medicago truncatula.

The size of leaf and seed organs, determined by the interplay of cell proliferation and expansion, is closely related to the final yield and quality of forage and crops. Yet the cellular and molecular mechanisms underlying organ size modulation remain poorly understood, especially in legumes. Here, MINI ORGAN1 (MIO1), which encodes an F-box protein SMALL LEAF AND BUSHY1 (SLB1) recently reported to control lateral branching in Medicago truncatula, was identified as a key regulator of organ size. We show that loss-of-function of MIO1/SLB1 severely reduced organ size. Conversely, plants overexpressing MIO1/SLB1 had enlarged organs. Cellular analysis revealed that MIO1/SLB1 controlled organ size mainly by modulating primary cell proliferation during the early stages of leaf development. Biochemical analysis revealed that MIO1/SLB1 could form part of SKP1/Cullin/F-box (SCF) E3 ubiquitin ligase complex, to target BIG SEEDS1 (BS1), a repressor of primary cell division, for degradation. Interestingly, we found that MIO1/SLB1 also played a key role in pulvinus development and leaf movement by modulating cell proliferation of the pulvinus as leaves developed. Our study not only demonstrates a conserved role of MIO1/SLB1 in the control of organ size in legumes, but also sheds light on the novel function of MIO1/SLB1 in leaf movement.

The WOX family transcriptional regulator SlLAM1 controls compound leaf and floral organ development in Solanum lycopersicum.

Plant-specific WOX family transcription factors play important roles ranging from embryogenesis to lateral organ development. The WOX1 transcription factors, which belong to the modern clade of the WOX family, are known to regulate outgrowth of the leaf blade specifically in the mediolateral axis; however, the role of WOX1 in compound leaf development remains unknown. Phylogenetic analysis of the whole WOX family in tomato (Solanum lycopersicum) indicates that there are 10 members that represent the modern, intermediate, and ancient clades. Using phylogenetic analysis and a reverse genetic approach, in this study we identified SlLAM1 in the modern clade and examined its function and tissue-specific expression pattern. We found that knocking out SlLAM1 via CRISPR/Cas9-mediated genome editing led to narrow leaves and a reduced number of secondary leaflets. Overexpression of tomato SlLAM1 could rescue the defects of the tobacco lam1 mutant. Anatomical and transcriptomic analyses demonstrated that floral organ development, fruit size, secondary leaflet initiation, and leaf complexity were altered due to loss-of-function of SlLAM1. These findings demonstrate that tomato SlLAM1 plays an important role in the regulation of secondary leaflet initiation, in addition to its conserved function in blade expansion.