Screening of Soybean Genotypes for Waterlogging Stress Tolerance and Understanding the Physiological Mechanisms

Waterlogging is a common form of abiotic stress that severely impedes global soybean production. Targeting this issue, an experiment was carried out at Sher-e-Bangla Agricultural University during August–November 2019 to screen out the waterlogging tolerance and yield performances of selected soybean genotypes. The experiment was laid out in a completely randomized design (CRD) with three replications consisting of 2 water levels (control and waterlogging) and 12 genotypes (Sohag, BARI Soybean-5, BINAsoybean-1, BINAsoybean-2, BINAsoybean-3, BINAsoybean-5, BINAsoybean-6, SGB-1, SGB-3, SGB-4, SGB-5, and GC-840). On the 15th day after sowing, plants were exposed to waterlogging for 12 days. Waterlogging remarkably declined the growth and yield of all the soybean genotypes compared to control. Reduced plant height, relative water content, above-ground fresh and dry weight, SPAD value, leaf area, number of leaves, branches, pods, seeds pod−1, 100-seed weight, and seed yield plant−1 were observed under waterlogging stress. Conversely, mortality rate and electrolyte leakage were increased under the same condition. The waterlogged plants showed delayed flowering and maturity compared with the control plants. However, among the 12 genotypes, Sohag, BARI Soybean-5, GC-840, BINAsoybean-1, and BINAsoybean-2 showed better waterlogging tolerance. These genotypes showed a greater number of adventitious roots in the base of their stem, which probably helped plants to thrive under waterlogging conditions.

Characterization of Squamosa-Promoter Binding Protein-Box Family Genes Reveals the Critical Role of MsSPL20 in Alfalfa Flowering Time Regulation

SQUAMOSA Promoter-binding protein-Like (SPL) genes affect a broad range of plant biological processes and show potential application in crop improvement by genetic modification. As the most widely planted forage crop in the world, biomass and abiotic stresses tolerance are important breeding targets for alfalfa (Medicago sativa L.). Nevertheless, the systematic analysis of SPL genes in alfalfa genome remains lacking. In the present study, we characterized 22 putative non-redundant SPL genes in alfalfa genome and uncovered the abundant structural variation among MsSPL genes. The phylogenetic analysis of plant SPL proteins separated them into 10 clades and clade J was an alfalfa-specific clade, suggesting SPL genes in alfalfa might have experienced gene duplication and functional differentiation within the genome. Meanwhile, 11 MsSPL genes with perfect matches to miRNA response elements (MREs) could be degraded by miR156, and the cleavage sites were gene specific. In addition, we investigated the temporal and spatial expression patterns of MsSPL genes and their expression patterns in response to multiple treatments, characterizing candidate SPL genes in alfalfa development and abiotic stress tolerant regulation. More importantly, overexpression of the alfalfa-specific SPL gene (MsSPL20) showed stable delayed flowering time, as well as increased biomass. Further studies indicated that MsSPL20 delayed flowering time by regulating the expression of genes involved in floret development, including HD3A, FTIP1, TEM1, and HST1. Together, our findings provide valuable information for future research and utilization of SPL genes in alfalfa and elucidate a possibly alfalfa-specific flowering time regulation, thereby supplying candidate genes for alfalfa molecular-assisted breeding.

Kochia (Bassia Scoparia) Growth and Fecundity As Influenced By Crop Competition and Weed Density

Kochia [Bassia scoparia (L.) A. J. Scott] represents one the most troublesome weeds in crop production systems in the North American Great Plains. The development of herbicide-resistant B. scoparia populations further exacerbated this problem. More ecologically driven approaches to its control are necessary. This study examined the competitive effects of four crops (sugar beet, soybean, barley, and corn) in combination with B. scoparia densities (3, 13, 24, 47, 94, and 188 plants m-2) on B. scoparia development and seed production across 2 years. Corn and barley had the greatest impact on B. scoparia growth and fecundity. B. scoparia biomass was 87 and 82% lower and seed production was 98 and 96% lower (p<0.001) in corn and barley, respectively, relative to fallow. Corn had greatest effect in reducing B. scoparia biomass and seed production. Barley had greatest effect in delaying B. scoparia flowering which occurred 113 days after B. scoparia emergence (p<0.001). Soybean and sugar beet had the least effect reducing B. scoparia biomass by 70 and 65% and seed production by 84 and 80% (p<0.001), respectively, relative to fallow. Increasing B. scoparia densities resulted in reductions in B. scoparia width, number of primary branches, biomass plant-1, and seeds plant-1 but increased B. scoparia height, biomass m-2, and seeds m-2 (p<0.001) under all cropping treatments except corn. Barley represents the greatest opportunity to impact B. scoparia through reduced fecundity and delayed flowering, with the latter providing a window of opportunity for post-harvest control. The effects observed here were isolated from differences in herbicide practices that are associated with each of these crops, differences that have a dramatic effect on B. scoparia in their own right.

Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

Fine Mapping and Analysis of Candidate Genes for qFT7.1, a Major Quantitative Trait Locus Controlling Flowering Time in Brassica Rapa L

In Brassica rapa, flowering time (FT) is an important agronomic trait that affects the yield, quality, and adaption. FT a complicated trait that is regulated by many genes and is affected greatly by the environment. In this study, a chromosome segment substitution line (CSSL), CSSL16, was selected that showed later flowering than the recurrent parent, rapid-cycling inbred line of B. rapa (RcBr). Using Bulked Segregant RNA sequencing, we identified a late flowering quantitative trait locus (QTL), designated as qFT7.1, on chromosome A07 based on a secondary-F2 population derived from the cross between CSSL16 and RcBr. qFT7.1 was further validated by conventional QTL mapping. This QTL explained 39.9% (logarithm of odds = 32.2) of the phenotypic variations and was fine mapped to a 56.4-kb interval using recombinant analysis. Expression analysis suggests that BraA07g018240.3C, which is homologous with ATC (encoding Arabidopsis thaliana CENTRORADIALIS homologue), a gene for delayed flowering in Arabidopsis as the most promising candidate gene. Sequence analysis demonstrated that two synonymous mutations existed in the coding region and numerous bases replacements existed in promoter region between BraA07g018240.3C from CSSL16 and RcBr. The results will increase our knowledge related to the molecular mechanism of late flowering in B. rapa, and lay a solid foundation for the breeding of late bolting in B. rapa.

Pontin/Reptin-associated complexes differentially impact plant development and viral pathology

Pontin and Reptin are essential eukaryotic AAA+ ATPases that work together in several multiprotein complexes, contributing to chromatin remodeling and TARGET OF RAPAMCYIN (TOR) kinase complex assembly, among other functions. Null alleles of pontin or reptin are gametophyte lethal in plants, which has hindered studies of their crucial roles in plant biology. Here, we used virus-induced gene silencing (VIGS) to interrogate the functions of Pontin and Reptin in plant growth and physiology, focusing on Nicotiana benthamiana, a model species for the agriculturally significant Solanaceae family. Silencing either Pontin or Reptin caused pleiotropic developmental and physiological reprogramming, including aberrant leaf shape, reduced apical growth, delayed flowering, increased branching, chlorosis, and decreased spread of the RNA viruses Tobacco mosaic virus (TMV) and Potato virus X (PVX). To dissect these pleiotropic phenotypes, we took a comparative approach and silenced expression of key genes that encode subunits of each of the major Pontin/Reptin-associated chromatin remodeling or TOR complexes (INO80, SWR-C/PIE1, TIP60, TOR, and TELO2). We found that many of the pontin/reptin phenotypes could be attributed specifically to disruption of one of these complexes, with tip60 and tor knockdown plants each phenocopying a large subset of pontin/reptin phenotypes. We conclude that Pontin/Reptin complexes are crucial for proper plant development, physiology, and stress responses, highlighting the multifaceted roles these conserved enzymes have evolved in eukaryotic cells.

A Wheat TaTOE1-B1 Transcript TaTOE1-B1-3 Can Delay the Flowering Time of Transgenic Arabidopsis

Flowering time is one of the most important agronomic traits in wheat production. A proper flowering time might contribute to the reduction or avoidance of biotic and abiotic stresses, adjust plant architecture, and affect the yield and quality of grain. In this study, TaTOE1-B1 in wheat produced three transcripts (TaTOE1-B1-1, TaTOE1-B1-2, and TaTOE1-B1-3) by alternative splicing. Compared to the longest transcript, TaTOE1-B1-1, TaTOE1-B1-3 has a deletion in the sixth exon (1219–1264 bp). Under long-day conditions, the heterologous overexpression of the TaTOE1-B1-3 gene delayed flowering, prolonged the vegetative growth time, and enlarged the vegetative body of Arabidopsis, but that of TaTOE1-B1-1 did not. As typical AP2 family members, TaTOE1-B1-1 and TaTOE1-B1-3 are mainly located in the nucleus and have transcriptional activation activities; the transcriptional activation region of TaTOE1-B1-3 is located in the C-terminal. In TaTOE1-B1-3 overexpression lines, the expression of flowering-related AtFT and AtSOC1 genes is significantly downregulated. In addition, this study confirms the protein–protein interaction between TaTOE1-B1-3 and TaPIFI, which may play an important role in flowering inhibition. These results provide a theoretical basis for the precise regulation of wheat flowering time.

GrTCP11, a Cotton TCP Transcription Factor, Inhibits Root Hair Elongation by Down-Regulating Jasmonic Acid Pathway in Arabidopsis thaliana

TCP transcription factors play important roles in diverse aspects of plant development as transcriptional activators or repressors. However, the functional mechanisms of TCPs are not well understood, especially in cotton fibers. Here, we identified a total of 37 non-redundant TCP proteins from the diploid cotton (Gossypium raimondii), which showed great diversity in the expression profile. GrTCP11, an ortholog of AtTCP11, was preferentially expressed in cotton anthers and during fiber initiation and secondary cell wall synthesis stages. Overexpression of GrTCP11 in Arabidopsis thaliana reduced root hair length and delayed flowering. It was found that GrTCP11 negatively regulated genes involved in jasmonic acid (JA) biosynthesis and response, such as AtLOX4, AtAOS, AtAOC1, AtAOC3, AtJAZ1, AtJAZ2, AtMYC2, and AtERF1, which resulted in a decrease in JA concentration in the overexpressed transgenic lines. As with the JA-deficient mutant dde2-2, the transgenic line 4-1 was insensitive to 50 μM methyl jasmonate, compared with the wild-type plants. The results suggest that GrTCP11 may be an important transcription factor for cotton fiber development, by negatively regulating JA biosynthesis and response.

Drought and Darkness during Long-Term Simulated Shipping Delay Post-Shipping Flowering of Phalaenopsis Sogo Yukidian ‘V3’

We investigated the relationship between simulated shipping (SS) without watering or light and post-shipping growth and flowering of Phalaenopsis Sogo Yukidian ‘V3’. Two experimental environments were created: a low-temperature chamber for simulated shipping and a growth chamber for simulated finishing at the destination. Plants from both the control and treatment groups were moved from the low-temperature chamber to the growth chamber after the end of the simulated shipping. Control plants received continuous light and regular irrigation; plants in the treatment group were placed in the low-temperature chamber under light (LSS) or dark (DSS) conditions for 10, 20, 30, 40, or 50 days, without irrigation. Once DSS duration exceeded 40 days, the leaf-yellowing rate increased rapidly. Chlorophyll content decreased from day 10 to 30 of DSS and slightly increased in LSS and DSS over 40 days. The photochemical reflectance index decreased with the SS duration. The maximum quantum yield PSII photochemistry (Fv/Fm) values sharply decreased after the end of SS; after 40 days, neither LSS nor DSS plants recovered to the normal range. In the same SS duration, the number of days to spiking was delayed in the DSS. In addition, the number of days to spiking was delayed, owing to the longer SS duration. LSS for 50 days induced early flowering, as in the control group, but lowered flower quality. The results demonstrate that drought stress from long-term shipping (>40 days) delayed flowering. In particular, DSS delayed flowering more than LSS due to the decrease in chlorophyll content and the reduction in carbohydrates through respiration.

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar ‘Kanai Sensei’. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.