Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

Phytochemical investigations and biological work on aerial parts and roots of Trigonella polycerata.

The purpose of this study was to purify the phytoconstituents and to explore the antibacterial, antifungal, phytotoxic and cytotoxic potential of dichloromethane and methanol extracts of aerial and root parts of Trigonella polycerata. The phytochemical study on methanol extract of aerial parts of the plant led to the isolation and purification of seven compounds that were identified as 3,4-dimethoxycinnamaldehyde, Trigocoumarin, 6,7,8-trimethoxycoumarin, Penduletin, 5-hydroxy-3,6,7,4´-tetramethoxyflavone, 3,5,7-trihydroxy-6,4-dimethoxyflavone and 5-hydroxy-4,7-dimethoxyflavone. These structures were elucidated by interpretation of EI-MS and NMR spectral data. The plant aerial parts methanol extract (TPAM) demonstrated higher antibacterial (78.99%), phytotoxic (85% growth regulation at 1000µg/mL) and cytotoxic activities (LD50: 45.643µg/mL). While the methanol root extract (TPRM) was highly active against bacteria's; Salmonella typhi (71.56%), Staphylococcus aureus (70.15%), Escherichia coli (69%), fungi like Candida albicans (70.21%) and moderately active against Brine shrimp larvae (LD50: 125.663µg/mL). The dichloromethane aerial (TPAD) and root (TPRD) extracts exhibited significant antibacterial (78.03% and 50.21% inhibitions respectively) and phytotoxic (55% growth regulation at 1000µg/mL) potential. Only TPAD indicated the best inhibition against fungi; Aspergillus flavus (75.31%) and moderate inhibition against Microsporum canis (42.21%). This phytochemical and biological work is the first time reported in Trigonella polycerata.

OsFTL4, an FT-like Gene, Regulates Flowering Time and Drought Tolerance in Rice (Oryza sativa L.).

The initiation of flowering in cereals is a critical process influenced by environmental and endogenous signals. Flowering Locus T-like (FT-like) genes encode the main signals for flowering. Of the 13 FT-like genes in the rice genome, Hd3a/OsFTL2 and RFT1/OsFTL3 have been extensively studied and revealed to be critical for flowering. In this study, a rice FT-like gene, OsFTL4, was functionally characterized. Specifically, osftl4 mutants were generated using a CRISPR/Cas9 system. Compared with the wild-type control (Guangluai 4), the osftl4-1 and osftl4-2 mutants flowered 9.6 and 5.8 days earlier under natural long-day and short-day conditions, respectively. Additionally, OsFTL4 was mainly expressed in the vascular tissue, with the resulting OsFTL4 protein localized in both the nucleus and cytoplasm. Furthermore, OsFTL4 was observed to compete with Hd3a for the interaction with multiple 14-3-3 proteins. An analysis of the effects of simulated drought stress suggested that silencing OsFTL4 enhances drought tolerance by decreasing stomatal conductance and water loss. These results indicate that OsFTL4 helps integrate the flowering process and the drought response in rice.

Methylation and expression of rice NLR genes after low temperature stress.

Nucleotide-binding leucine-rich repeat receptors (NLRs) are included in most plant disease resistance proteins. Some NLR proteins have been revealed to be induced by the invasion of plant pathogens. DNA methylation is required for adaption to adversity and proper regulation of gene expression in plants. Low temperature stress (LTS) is a restriction factor in rice growth, development and production. Here, we report the methylation and expression of NLR genes in two rice cultivars, i.e., 9311 (an indica rice cultivar sensitive to LTS), and P427 (a japonica cultivar, tolerant to LTS), after LTS. We found that the rice NLR genes were heavily methylated at CG sites at room temperature and low temperature in 9311 and P427, and many rice NLR genes showed DNA methylation alteration after LTS. A great number of rice NLR genes were observed to be responsive to LTS at the transcriptional level. Our observation suggests that the alteration of expression of rice NLR genes was similar but their change in DNA methylation was dynamic between the two rice cultivars after LTS. We identified that more P427 NLR genes reacted to LTS than those of 9311 at the methylation and transcriptional level. The results in this study will be useful for further understanding the transcriptional regulation and potential functions of rice NLR genes.

Fine Mapping of qTGW7b, a Minor Effect QTL for Grain Weight in Rice (Oryza sativa L.).

Grain weight is a key trait that determines rice quality and yield, and it is primarily controlled by quantitative trait loci (QTL). Recently, attention has been paid to minor QTLs. A minor effect QTL qTGW7 that controls grain weight was previously identified in a set of chromosomal fragment substitution lines (CSSLs) derived from Nipponbare (NPB)/93-11. Compared to NPB, the single segment substitution line (SSSL) N83 carrying the qTGW7 introgression exhibited an increase in grain length and width and a 4.5% increase in grain weight. Meanwhile, N83 was backcrossed to NPB to create a separating population, qTGW7b, a QTL distinct from qTGW7, which was detected between markers G31 and G32. Twelve near-isogenic lines (NILs) from the BC9F3 population and progeny of five NILs from the BC9F3:4 population were genotyped and phenotyped, resulting in the fine mapping of the minor effect QTL qTGW7b to the approximately 86.2-kb region between markers G72 and G32. Further sequence comparisons and expression analysis confirmed that five genes, including Os07g39370, Os07g39430, Os07g39440, Os07g39450, and Os07g39480, were considered as the candidate genes underlying qTGW7b. These results provide a crucial foundation for further cloning of qTGW7b and molecular breeding design in rice.

Transcriptome Profiling Reveals Role of MicroRNAs and Their Targeted Genes during Adventitious Root Formation in Dark-Pretreated Micro-Shoot Cuttings of Tetraploid Robinia pseudoacacia L.

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L-1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.

InDel Marker Based Estimation of Multi-Gene Allele Contribution and Genetic Variations for Grain Size and Weight in Rice (Oryza sativa L.).

The market success of any rice cultivar is exceedingly dependent on its grain appearance, as well as its grain yield, which define its demand by consumers as well as growers. The present study was undertaken to explore the contribution of nine major genes, qPE9~1, GW2, SLG7, GW5, GS3, GS7, GW8, GS5, and GS2, in regulating four size and weight related traits, i.e., grain length (GL), grain width (GW), grain thickness (GT), and thousand grain weight (TGW) in 204 diverse rice germplasms using Insertion/Deletion (InDel) markers. The studied germplasm displayed wide-ranging variability in the four studied traits. Except for three genes, all six genes showed considerable association with these traits with varying strengths. Whole germplasm of 204 genotypes could be categorized into three major clusters with different grain sizes and weights that could be utilized in rice breeding programs where grain appearance and weight are under consideration. The study revealed that TGW was 24.9% influenced by GL, 37.4% influenced by GW, and 49.1% influenced by GT. Hence, assuming the trend of trait selection, i.e., GT > GW > GL, for improving TGW in the rice yield enhancement programs. The InDel markers successfully identified a total of 38 alleles, out of which 27 alleles were major and were found in more than 20 genotypes. GL was associated with four genes (GS3, GS7, GW8, and GS2). GT was also found to be regulated by four different genes (GS3, GS7, GW8, and GS2) out of the nine studied genes. GW was found to be under the control of three studied genes (GW5, GW8, and GS2), whereas TGW was found to be under the influence of four genes (SLG7, GW5, GW8, and GS5) in the germplasm under study. The Unweighted Pair Group Method with Arithmetic means (UPGMA) tree based on the studied InDel marker loci segregated the whole germplasm into three distinct clusters with dissimilar grain sizes and weights. A two-dimensional scatter plot constructed using Principal Coordinate Analysis (PCoA) based on InDel markers further separated the 204 rice germplasms into four sub-populations with prominent demarcations of extra-long, long, medium, and short grain type germplasms that can be utilized in breeding programs accordingly. The present study could help rice breeders to select a suitable InDel marker and in formulation of breeding strategies for improving grain appearance, as well as weight, to develop rice varieties to compete international market demands with higher yield returns. This study also confirms the efficient application of InDel markers in studying diverse types of rice germplasm, allelic frequencies, multiple-gene allele contributions, marker-trait associations, and genetic variations that can be explored further.