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Abstract 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.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

In legumes, many endogenous and environmental factors affect root nodule formation through several key genes, and the regulation details of the nodulation signaling pathway are yet to be fully understood. This study investigated the potential roles of terpenoids and terpene biosynthesis genes on root nodule formation in Glycine max. We characterized six terpenoid synthesis genes from Salvia officinalis by overexpressing SoTPS6, SoNEOD, SoLINS, SoSABS, SoGPS, and SoCINS in soybean hairy roots and evaluating root growth and nodulation, and the expression of strigolactone (SL) biosynthesis and early nodulation genes. Interestingly, overexpression of some of the terpenoid and terpene genes increased nodule numbers, nodule and root fresh weight, and root length, while others inhibited these phenotypes. These results suggest the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. This study provides novel insights into epistatic interactions between terpenoids, root development, and nodulation in soybean root biology and open new avenues for soybean research.

Root nodule formation in many leguminous plants is known to be affected by endogen ous and exogenous factors that affect formation, development, and longevity of nodules in roots. Therefore, it is important to understand the role of the genes which are involved in the regulation of the nodulation signaling pathway. This study aimed to investigate the effect of terpenoids and terpene biosynthesis genes on root nodule formation in Glycine max. The study aimed to clarify not only the impact of over-expressing five terpene synthesis genes isolated from G. max and Salvia guaranitica on soybean nodulation signaling pathway, but also on the strigolactones pathway. The obtained results revealed that the over expression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes enhanced the root nodule numbers, fresh weight of nodules, root, and root length. Moreover, the terpene content in the transgenic G. max hairy roots was estimated. The results explored that the monoterpenes, sesquiterpenes and diterpenes were significantly increased in transgenic soybean hairy roots in comparison with the control. Our results indicate the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. The study provides novel insights for understanding the epistatic relationship between terpenoids, root development, and nodulation in soybean.

Abstract Background Two crossing techniques for hybridization of chickpea have been reported and include pollination after emasculation and pollination without emasculation. Success of crossing with emasculation varied from 5 to 17%; while the success rate varied from 20 to 50% by pollination without emasculation. The important reason for the low success rate of the two procedures could be lack of detailed information on the flowering stages chosen for crossing together with the environment where plants grow. Results We describe a comprehensive method for chickpea crossing where two genotypes, ICCV96029 as female and PI503023 as male parent were used. Leaf shape and seed size were used as morphological markers to select hybrids. For crossing, incision was made along the central line of the keel petal for the removal of anthers and to expose the stigma for placement of pollen from donor parent on its surface. After pollination, style was inserted back gently inside the keel petal and covered by wing petals and standard petals to make a natural sac which prevents drying of internal organs. Alternatively, if the conditions are favorable there is no need to protect the pollinated flower and therefore petal removal method for cross-pollination can be used. Our method showed around 78% crossing success rate which is much higher than the previous results. Conclusions We have shown that the crossing by keel petal incision or petal removal is an effective approach which significantly increases the crossing success rate. Furthermore, our detailed method shows that the flowering stage, selection of parents and temperature play crucial roles in crossing success.

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

Molecular genetic studies suggest that FLORICA LILA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisuni sativum and Lotus japonicus. To further understand the role of FLOILFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leafleti (sgll), from the model legume Medicago truncatula. The sgll mutants exhibited strong defects in compound leaf development; all adult leaves in sgll mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgll mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGLTI revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgll mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGLI genomic sequence completely rescued floral defects of Ify mutants, it failed to alter the simple leaf structure of the A rabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula. [ABSTRACT FROM AUTHOR]