Numbers on branches indicate bootstrap percentages for 1000 replicates. (b) Subcellular localization of MtDMP8-RFP and MtDMP9-RFP proteins in Arabidopsis leaf protoplasts; pm-GFP was used as a plasma ...membrane marker. Consistent with this prediction, both proteins colocalized with the PIP2A (At3g53420)-based plasma membrane marker pm-GFP (Zhu et al., 2020) when MtDMP8 and MtDMP9 were transiently expressed as red fluorescent protein (RFP) fusions in Arabidopsis leaf protoplasts (Figure 1b). The haploid plants were morphologically similar to the female parent A17 (Figure 1k). ...the simultaneous inactivation of MtDMP8 and MtDMP9 can trigger in vivo maternal haploid induction in M. truncatula.
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 WUS-box 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.
Plant height is an important agronomic trait that is closely related to biomass yield and crop production. Despite legumes comprise one of the largest monophyletic families that are second only to ...grasses in terms of economic and nutritional values, due to an ancient genome duplication event, most legume plants have complex genomes, thus the molecular mechanisms that determine plant height are less known in legumes. Here, we report the identification and characterization of
MAIN STEM DWARF1
(
MSD1
), which is required for the plant height in the model legume
Medicago truncatula
. Loss of function of
MSD1
leads to severely reduced main stem height but normal lateral branch elongation in
M. truncatula.
Histological analysis revealed that the
msd1-1
main stem has shorter internodes with reduced cell size and number compared with the wild type, indicating that
MSD1
affects cell elongation and cell proliferation.
MSD1
encodes a putative GA 20-oxidase that is expressed at significantly higher levels in the main shoot apex than in the lateral shoot apices, suggesting that
MSD1
expression is associated with its effect on the main stem elongation. UPLC-MS/MS analysis showed that GA
9
and GA
4
, two identified products of the GA 20-oxidase, were severely reduced in
msd1-1
, and the dwarf phenotype of
msd1-1
could be rescued by supplementation with gibberellic acid GA
3
, confirming that
MSD1
functions as a biologically active GA 20-oxidase. Moreover, we found that disruption of either
MtGA20ox7
or
MtGA20ox8
, homologs of
MSD1
, has little effects on the elongation of the main stem, while the
msd1-1 mtga20ox7-1 mtga20ox8
triple mutants exhibits a severe short main shoot and lateral branches, as well as reduced leaf size, suggesting that
MSD1
and its homologs
MtGA20ox7
and
MtGA20ox8
, redundantly regulate
M. truncatula
shoot elongation and leaf development. Taken together, our findings demonstrate the molecular mechanism of
MSD1
-mediated regulation of main stem elongation in
M. truncatula
and provide insights into understanding the functional diversity of GA 20-oxidases in optimizing plant architecture in legumes.
Two ABCG transporters in Medicago truncatula collaboratively regulate flower cuticle secretion and control stigma exsertion by affecting physical contact between floral organs.
Abstract
Stigma ...exsertion is an important agricultural trait that facilitates the application of heterosis in crop breeding. Although several quantitative trait loci associated with stigma exsertion have been fine-mapped or cloned, the underlying genetic basis, particularly in legumes, remains unclear. In this study, we identified and characterized the exserted stigma mutant stigma exsertion1 (sge1) in the model legume Medicago truncatula. The exserted stigma phenotype of sge1 is mainly caused by physical interaction between floral organs, in which normal petal and stamen elongation are inhibited due to flower cuticle defects. SGE1 encodes an ATP-binding cassette G (ABCG) transporter that plays a critical role in regulating floral cutin and wax secretion in M. truncatula. SGE1 physically interacts with another half-size transporter, MtABCG13, to form a functional heterodimer. Mutation of MtABCG13 results in flower cuticle defects similar to those in sge1 as well as stigma exsertion, indicating that SGE1 and MtABCG13 are indispensable for flower cuticle secretion and collaboratively control stigma exsertion in M. truncatula. Our findings reveal novel functions for ABCG transporters in determining stigma exsertion by affecting the physical interactions of floral organs, providing insight into the molecular mechanism underlying stigma exsertion in leguminous plants with complex zygomorphic flowers.
Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral ...transition in sorghum is poorly understood.
Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches.
Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum.
SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.
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.
Robust single-stranded DNA (ssDNA) microarrays are created by attaching amine-modified oligonucleotides to a monolayer of poly(L-glutamic acid) (pGlu) that is electrostatically adsorbed onto a ...chemically modified gold thin film. This surface attachment chemistry methodology is first characterized with a combination of polarization-modulation Fourier transform infrared (PM-FTIR) spectroscopy and surface plasmon resonance (SPR) angle shift measurements. SPR imaging (SPRI) measurements of these ssDNA microarrays are then used to study two surface bioaffinity interactions: (i) the quantitative hybridization adsorption of complementary ssDNA onto mixed ssDNA microarray elements and (ii) the adsorption of single-stranded binding protein (SSB) onto fully and partially hybridized DNA microarray elements. The Langmuir adsorption coefficient (K(Ads)) of SSB binding to ssDNA was determined to be (5.5 +/- 0.4) x 10(9) M(-1).
Plasmonics is generally divided into two categories: surface plasmon resonance (SPR) of electromagnetic modes propagating along a (noble) metal/dielectric interface and localized SPRs (LSPRs) on ...nanoscopic metallic structures (particles, rods, shells, holes, etc.). Both optical transducer concepts can be combined with and integrated in microfluidic devices for biomolecular analyte detections, with the benefits of small foot-print for point-of-care detection, low-cost for one-time disposal, and ease of being integrated into an array format. The key technologies in such integration include the plasmonic chip, microfluidic channel fabrication, surface bio-functionalization, and selection of the detection scheme, which are selected according to the specifics of the targeting analytes. This paper demonstrates a few examples of the many versions of how to combine plasmonics and integrated microfluidics, using different plasmonic generation mechanisms for different analyte detections. One example is a DNA sensor array using a gold film as substrate and surface plasmon fluorescence spectroscopy and microscopy as the transduction method. This is then compared to grating-coupled SPR for poly(ethylene glycol) thiol interaction detected by angle interrogation, gold nanohole based LSPR chip for biotin-strepavidin detection by wavelength shift, and gold nanoholes/nanopillars for the detection of prostate specific antigen by quantum dot labels excited by the LSPR. Our experimental results exemplified that the plasmonic integrated microfluidics is a promising tool for understanding the biomolecular interactions and molecular recognition process as well as biosensing, especially for on-site or point-of-care diagnostics.
Switchgrass (Panicum virgatum L.), a perennial warm season bunchgrass native to North America, has been a target in the U.S. as a renewable bioenergy crop because of its ability to produce moderate ...to high biomass yield on marginal soils. Delaying flowering can increase vegetative biomass production by allowing prolonged growth before switching to the reproductive phase. Despite the identification of flowering time as a biomass trait in switchgrass, the molecular regulatory factors involved in controlling floral transition are poorly understood. Here we identified PvFT1, PvAPL1‐3 and PvSL1, 2 as key flowering regulators required from floral transition initiation to development of floral organs. PvFT1 expression in leaves is developmentally regulated peaking at the time of floral transition, and diurnally regulated with peak at approximately 2 h into the dark period. Ectopic expression of PvFT1 in Arabidopsis, Brachypodium and switchgrass led to extremely early flowering, and activation of FT downstream target genes, confirming that it is a strong activator of flowering in switchgrass. Ectopic expression of PvAPL1‐3 and PvSL1, 2 in Arabidopsis also activated early flowering with distinct floral organ phenotypes. Our results suggest that switchgrass has conserved flowering pathway regulators similar to Arabidopsis and rice.
Floral transition is an important biomass trait because longer vegetative phase results in the accumulation of more lignocellulosic biomass. Switchgrass is one of the dedicated biomass feedstocks in the U.S. but the control of its floral transition has not been studied at the molecular level. In this report, we identified PvFT1 as one of the switchgrass florigens together with three AP1‐like downstream targets and two SOC1‐like genes, which together comprise the switchgrass floral integrators similar to Arabidopsis and rice. This work lays the foundation for understanding the molecular mechanism of floral transition in switchgrass and its ultimate manipulation for improving biomass feedstock yield.
Alfalfa (
L.) is an important forage crop worldwide, but molecular genetics and breeding research in this species are hindered by its self-incompatibility (SI). Although the mechanisms underlying SI ...have been extensively studied in other plant families, SI in legumes, including alfalfa, remains poorly understood. Here, we determined that self-pollinated pollen tubes could germinate on the stigma of alfalfa, grow through the style, and reach the ovarian cavity, but the ovules collapsed ~48 h after self-pollination. A transcriptomic analysis of dissected pistils 24 h after self-pollination identified 941 differently expressed genes (DEGs), including 784 upregulated and 157 downregulated genes. A gene ontology (GO) analysis showed that the DEGs were highly enriched in functions associated with the regulation of pollen tube growth and pollen germination. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that pentose and glucuronate interconversion, plant hormone signal transduction, the spliceosome, and ribosomes might play important roles in SI. Our co-expression analysis showed that F-box proteins, serine/threonine protein kinases, calcium-dependent protein kinases (CDPKs), bHLHs, bZIPs, and MYB-related family proteins were likely involved in the SI response. Our study provides a catalog of candidate genes for further study to understand SI in alfalfa and related legumes.
PDF
