Genetic mapping of the wheat leaf rust resistance gene Lr2a and its importance in Canadian wheat cultivars.

KEY MESSAGE: Leaf rust resistance gene Lr2a was located to chromosome arm 2DS in three mapping populations, which will facilitate map-based cloning and marker-assisted selection of Lr2a in wheat breeding programs. Incorporating effective leaf rust resistance (Lr) genes into high-yielding wheat cultivars has been an efficient method of disease control. One of the most widely used genes in Canada is the multi-allelic resistance gene Lr2, with alleles Lr2a, Lr2b, Lr2c, and Lr2d. The Lr2a allele confers complete resistance to a large portion of the Puccinia triticina (Pt) population in Canada. In this study, Lr2a was genetically mapped in two doubled haploid populations developed from the crosses Superb/BW278 and Superb/86ISMN 2137, and an F2 population developed from the cross Chinese Spring/RL6016. Seedlings were tested with the Lr2a avirulent Pt races 74-2 MGBJ (Superb/BW278) and 12-3 MBDS (Superb/86ISMN 2137 and Chinese Spring/RL6016) in greenhouse assays and were genotyped with 90K wheat Infinium SNP and kompetitive allele-specific PCR (KASP) markers. Lr2a was mapped to a collinear position on chromosome arm 2DS in all three populations, within a 1.00 cM genetic interval between KASP markers kwm1620 and kwm1623. This corresponded to a 305 kb genomic region of chromosome 2D in Chinese Spring RefSeq v2.1. The KASP marker kwh740 was predictive of Lr2a in all mapping populations. A panel of 260 wheats were tested with three Pt isolates, which revealed that Lr2a is common in Canadian wheats. The KASP markers kwh740 and kwm1584 were highly associated with resistance at the Lr2 locus, while kwm1622 was slightly less correlated. Genetic mapping of the leaf rust resistance gene Lr2a and DNA markers developed here will facilitate its use in wheat breeding programs.

Modulation of Plant MicroRNA Expression: Its Potential Usability in Wheat (Triticum aestivum L.) Improvement.

Wheat, a crucial crop for the pursuit of food security, is faced with a plateauing yield projected to fall short of meeting the demands of the exponentially increasing human population. To raise global wheat productivity levels, strong efforts must be made to overcome the problems of (1) climate change-induced heat and drought stress and (2) the genotype-dependent amenability of wheat to tissue culture, which limits the success of recovering genetically engineered plants, especially in elite cultivars. Unfortunately, the mainstream approach of genetically engineering plant protein-coding genes may not be effective in solving these problems as it is difficult to map, annotate, functionally verify, and modulate all existing homeologs and paralogs within wheat's large, complex, allohexaploid genome. Additionally, the quantitative, multi-genic nature of most agronomically important traits furthers the complications faced by this approach. miRNAs are small, noncoding RNAs (sncRNAs) that repress gene expression at the post-transcriptional level, regulating various aspects of plant growth and development. They are gaining popularity as alternative targets of genetic engineering efforts for crop improvement due to their (1) highly conserved nature, which facilitates reasonable prediction of their gene targets and phenotypic effects under different expression levels, and (2) the capacity to target multiple genes simultaneously, making them suitable for enhancing complex and multigenic agronomic traits. In this mini-review, we will discuss the biogenesis, manipulation, and potential applications of plant miRNAs in improving wheat's yield, somatic embryogenesis, thermotolerance, and drought-tolerance in response to the problems of plateauing yield, genotype-dependent amenability to tissue culture, and susceptibility to climate change-induced heat and drought stress.  © His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food, 2023.

The genetics of Cannabis-genomic variations of key synthases and their effect on cannabinoid content.

Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalization in Canada, Cannabis is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allow for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single-nucleotide polymorphisms in the coding regions of cannabinoid synthases play an important role in determining plant chemotype. Deep understanding of how these variants affect enzyme activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profiles. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.

Genome editing in wheat microspores and haploid embryos mediated by delivery of ZFN proteins and cell-penetrating peptide complexes.

Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value-added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell-penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN-treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

Transgenerational response to stress in plants and its application for breeding.

A growing number of reports indicate that plants possess the ability to maintain a memory of stress exposure throughout their ontogenesis and even transmit it faithfully to the following generation. Some of the features of transgenerational memory include elevated genome instability, a higher tolerance to stress experienced by parents, and a cross-tolerance. Although the underlying molecular mechanisms of this phenomenon are not clear, a likely contributing factor is the absence of full-scale reprogramming of the epigenetic landscape during gametogenesis; therefore, epigenetic marks can occasionally escape the reprogramming process and can be passed on to the progeny. To date, it is not entirely clear which part of the epigenetic landscape is more likely to escape the reprogramming events, and whether such a process is random or directed and sequence specific. The identification of specific epigenetic marks associated with specific stressors would allow generation of stress-tolerant plants through the recently discovered techniques for precision epigenome engineering. The engineered DNA-binding domains (e.g. ZF, TALE, and dCas9) fused to particular chromatin modifiers would make it possible to target epigenetic modifications to the selected loci, probably allowing stress tolerance to be achieved in the progeny. This approach, termed epigenetic breeding, along with other methods has great potential to be used for both the assessment of the propagation of epigenetic marks across generations and trait improvement in plants. In this communication, we provide a short overview of recent reports demonstrating a transgenerational response to stress in plants, and discuss the underlying potential molecular mechanisms of this phenomenon and its use for plant biotechnology applications.

Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.

Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.

Genome stability in the uvh6 mutant of Arabidopsis thaliana.

Plant XPD homolog UVH6 is the protein involved in the repair of strand breaks, and the excision repair and uvh6 mutant is not impaired in transgenerational increase in HRF. While analyzing the transgenerational response to stress in plants, we found that the promoter and gene body of Arabidopsis thaliana (Arabidopsis) XPD homolog UVH6 underwent hypomethylation and showed an increase in the level of transcript. Here, we analyzed the mutant of this gene, uvh6-1, by crossing it to two different reporter lines: one which allows for analysis of homologous recombination frequency (HRF) and another which makes it possible to analyze the frequency of point mutations. We observed that uvh6-1 plants exhibited lower rate of spontaneous homologous recombination but higher frequencies of spontaneous point mutations. The analysis of strand breaks using ROPS and Comet assays showed that the mutant had a much higher level of strand breaks at non-induced conditions. Exposure to stresses such as UVC, heat, cold, flood and drought showed that the mutant was not impaired in an increase in somatic HRF. The analysis of spontaneous HRF in the progeny of control plants compared to that of the progeny of stressed plants demonstrated that uvh6-1 was mildly affected in response to temperature, UV and drought. Our data suggest that UVH6 may be involved in the repair of strand breaks and excision repair, but it is unlikely that UVH6 is required for transgenerational increase in HRF.

Foliar application of plant-derived peptides decreases the severity of leaf rust (Puccinia triticina) infection in bread wheat (Triticum aestivum L.).

BACKGROUND: Screening and developing novel antifungal agents with minimal environmental impact are needed to maintain and increase crop production, which is constantly threatened by various pathogens. Small peptides with antimicrobial and antifungal activities have been known to play an important role in plant defense both at the pathogen level by suppressing its growth and proliferation as well as at the host level through activation or priming of the plant's immune system for a faster, more robust response against fungi. Rust fungi (Pucciniales) are plant pathogens that can infect key crops and overcome resistance genes introduced in elite wheat cultivars. RESULTS: We performed an in vitro screening of 18 peptides predominantly of plant origin with antifungal or antimicrobial activity for their ability to inhibit leaf rust (Puccinia triticina, CCDS-96-14-1 isolate) urediniospore germination. Nine peptides demonstrated significant fungicidal properties compared to the control. Foliar application of the top three candidates, ß-purothionin, Purothionin-α2 and Defensin-2, decreased the severity of leaf rust infection in wheat (Triticum aestivum L.) seedlings. Additionally, increased pathogen resistance was paralleled by elevated expression of defense-related genes. CONCLUSIONS: Identified antifungal peptides could potentially be engineered in the wheat genome to provide an alternative source of genetic resistance to leaf rust.

Genome stability of Arabidopsis atm, ku80 and rad51b mutants: somatic and transgenerational responses to stress.

DNA double-strand breaks (DSBs) can be repaired via two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). Our previous work showed that exposure to abiotic stresses resulted in an increase in point mutation frequency (PMF) and homologous recombination frequency (HRF), and these changes were heritable. We hypothesized that mutants impaired in DSB recognition and repair would also be deficient in somatic and transgenerational changes in PMF and HRF. To test this hypothesis, we analyzed the genome stability of the Arabidopsis thaliana mutants deficient in ATM (communication between DNA strand break recognition and the repair machinery), KU80 (deficient in NHEJ) and RAD51B (deficient in HR repair) genes. We found that all three mutants exhibited higher levels of DSBs. Plants impaired in ATM had a lower spontaneous PMF and HRF, whereas ku80 plants had higher frequencies. Plants impaired in RAD51B had a lower HRF. HRF in wild-type, atm and rad51b plants increased in response to several abiotic stressors, whereas it did not increase in ku80 plants. The progeny of stressed wild-type and ku80 plants exhibited an increase in HRF in response to all stresses, and the increase was higher in ku80 plants. The progeny of atm plants showed an increase in HRF only when the parental generation was exposed to cold or flood, whereas the progeny of rad51b plants completely lacked a transgenerational increase in HRF. Our experiments showed that mutants impaired in the recognition and repair of DSBs exhibited changes in the efficiency of DNA repair as reflected by changes in strand breaks, point mutation and HRF. They also showed that the HR RAD51B protein and the protein ATM that recognized damaged DNA might play an important role in transgenerational changes in HRF.

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation.

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

ddm1 plants are sensitive to methyl methane sulfonate and NaCl stresses and are deficient in DNA repair.

UNLABELLED: Plant response to stress includes changes in gene expression and chromatin structure. Our previous work showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational response to stress that included an increase in recombination frequency, cytosine methylation and stress tolerance. It can be hypothesized that changes in chromatin structure are important for an efficient stress response. To test this hypothesis, we analyzed the stress response of ddm1, a mutant impaired in DDM1, a member of the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes. We exposed Arabidopsis thaliana ddm1 mutants to methyl methane sulfonate (MMS) and NaCl and found that these plants were more sensitive. At the same time, ddm1 plants were similar to wild-type plants in sensitivity to temperature and bleomycin stresses. Direct comparison to met1 plants, deficient in maintenance methyltransferase MET1, showed higher sensitivity of ddm1 plants to NaCl. The level of DNA strand breaks upon exposure to MMS increased in wild-type plants but decreased in ddm1 plants. DNA methylation analysis showed that heterozygous ddm1/DDM1 plants had lower methylation as compared to fourth generation of homozygous ddm1/ddm1 plants. Exposure to MMS resulted in a decrease in methylation in wild-type plants and an increase in ddm1 plants. Finally, in vitro DNA excision repair assay showed lower capacity for ddm1 mutant. Our results provided a new example of a link between genetic genome stability and epigenetic genome stability. KEY MESSAGE: We demonstrate that heterozygous ddm1/DDM1 plants are more sensitive to stress and have more severe changes in methylation than homozygous ddm1/ddm1 plants.

Applications of CPPs in Genome Editing of Plants.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

Microsatellite instability in Arabidopsis increases with plant development.

Plant development consists of the initial phase of intensive cell division followed by continuous genome endoreduplication, cell growth, and elongation. The maintenance of genome stability under these conditions is the main task performed by DNA repair and genome surveillance mechanisms. Our previous work showed that the rate of homologous recombination repair in older plants decreases. We hypothesized that this age-dependent decrease in the recombination rate is paralleled with other changes in DNA repair capacity. Here, we analyzed microsatellite stability using transgenic Arabidopsis (Arabidopsis thaliana) plants that carry the nonfunctional ß-glucuronidase gene disrupted by microsatellite repeats. We found that microsatellite instability increased dramatically with plant age. We analyzed the contribution of various mechanisms to microsatellite instability, including replication errors and mistakes of DNA repair mechanisms such as mismatch repair, excision repair, and strand break repair. Analysis of total DNA polymerase activity using partially purified protein extracts showed an age-dependent decrease in activity and an increase in fidelity. Analysis of the steady-state RNA level of DNA replicative polymerases α, δ, Pol I-like A, and Pol I-like B and the expression of mutS homolog 2 (Msh2) and Msh6 showed an age-dependent decrease. An in vitro repair assay showed lower efficiency of nonhomologous end joining in older plants, paralleled by an increase in Ku70 gene expression. Thus, we assume that the more frequent involvement of nonhomologous end joining in strand break repair and the less efficient end-joining repair together with lower levels of mismatch repair activities may be the main contributors to the observed age-dependent increase in microsatellite instability.

Local infection with oilseed rape mosaic virus promotes genetic rearrangements in systemic Arabidopsis tissue.

We have previously shown that local infection of tobacco plants with tobacco mosaic virus (TMV) or oilseed rape mosaic virus (ORMV) results in a systemic increase in the homologous recombination frequency (HRF). Here, we analyzed what other changes in the genome are triggered by pathogen infection. For the analysis of HRF, mutation frequency (MF) and microsatellite instability (MI), we used three different transgenic Arabidopsis lines carrying ß-glucuronidase (GUS)-based substrates in their genome. We found that local infection of Arabidopsis with ORMV resulted in an increase of all three frequencies, albeit to differing degrees. The most prominent increase was observed in microsatellite instability. The increase in HRF was the lowest, although still statistically significant. The analysis of methylation of the 35S promoter and transgene expression showed that the greater instability of the transgene was not attributed to these changes. Strand breaks brought about a significant increase in non-treated tissues of infected plants. The expression of genes associated with various repair processes, such as KU70, RAD51, MSH2, DNA POL α and DNA POL δ, was also increased. To summarize, our data demonstrate that local ORMV infection destabilizes the genome in systemic tissues of Arabidopsis plants in various ways resulting in large rearrangements, point mutations and microsatellite instability.

Advances in Gene Editing of Haploid Tissues in Crops.

Emerging threats of climate change require the rapid development of improved varieties with a higher tolerance to abiotic and biotic factors. Despite the success of traditional agricultural practices, novel techniques for precise manipulation of the crop's genome are needed. Doubled haploid (DH) methods have been used for decades in major crops to fix desired alleles in elite backgrounds in a short time. DH plants are also widely used for mapping of the quantitative trait loci (QTLs), marker-assisted selection (MAS), genomic selection (GS), and hybrid production. Recent discoveries of genes responsible for haploid induction (HI) allowed engineering this trait through gene editing (GE) in non-inducer varieties of different crops. Direct editing of gametes or haploid embryos increases GE efficiency by generating null homozygous plants following chromosome doubling. Increased understanding of the underlying genetic mechanisms responsible for spontaneous chromosome doubling in haploid plants may allow transferring this trait to different elite varieties. Overall, further improvement in the efficiency of the DH technology combined with the optimized GE could accelerate breeding efforts of the major crops.

Chlorine ions but not sodium ions alter genome stability of Arabidopsis thaliana.

Various environmental stresses influence plant genome stability. Most of these stresses, such as ionizing radiation, heavy metals and organic chemicals, represent potent DNA-damaging agents. Here, we show that exposure to NaCl, the stress that is not thought to cause direct DNA damage, results in an increase in the level of strand breaks and homologous recombination rates (RRs) in Arabidopsis thaliana plants. The effect of salt stress on the RR was found to be primarily associated with Cl(-) ions, since exposure of plants to Na(2)SO(4) did not increase the RR, whereas exposure to MgCl(2) resulted in an increase. Changes in the number of strand breaks and in the RR were also paralleled by transcriptional activation of AtRad51 and down-regulation of AtKu70. The progeny of exposed plants exhibited higher RRs, higher expression of AtRad51, lower expression of AtKu70, higher tolerance to salt and methyl methane sulfate (MMS) stresses, as well as a higher increase in RR upon further exposure to stress. Our experiments showed that NaCl is a genotoxic stress that leads to somatic and transgenerational changes in recombination rates, and these changes are primarily triggered by exposure to Cl(-) ions.

Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress.

Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs.

Emerging Genome Engineering Tools in Crop Research and Breeding.

Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant's genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.

Development and Optimization of a Germination Assay and Long-Term Storage for Cannabis sativa Pollen.

Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, the bicellular nature of cannabis pollen was identified using DAPI (4',6-diamidino-2-phenylindole) staining. A pollen germination assay was developed to assess cannabis pollen viability and used to demonstrate that pollen collected from different principal growth stages exhibited differential longevity. Finally, a simple and efficient method that employs pollen combined with baked whole wheat flour and subsequent desiccation under vacuum was developed for the long-term cryopreservation of C. sativa pollen. Using this method, pollen viability was maintained in liquid nitrogen after four months, suggesting long-term preservation of cannabis pollen.

Transgenerational Response to Heat Stress in the Form of Differential Expression of Noncoding RNA Fragments in Brassica rapa Plants.

Epigenetic regulations in the form of changes in differential expression of noncoding RNAs (ncRNAs) are an essential mechanism of stress response in plants. Previously we showed that heat treatment in L. results in the differential processing and accumulation of ncRNA fragments (ncRFs) stemming from transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs). In this work, we analyzed whether ncRFs are differentially expressed in the progeny of heat-stressed plants. We found significant changes in the size of tRF reads and a significant decrease in the percentage of tRFs mapping to tRNA-Ala, tRNA-Arg, and tRNA-Tyr and an increase in tRFs mapping to tRNA-Asp. The enrichment analysis showed significant differences in processing of tRFs from tRNA, tRNA, tRNA, tRNA, tRNA, and tRNA isoacceptors. Analysis of potential targets of tRFs showed that they regulate brassinosteroid metabolism, the proton pump ATPase activity, the antiporter activity, the mRNA decay activity as well as nucleosome positioning and the epigenetic regulation of transgenerational response. Gene ontology term analysis of potential targets demonstrated a significant enrichment in tRFs that potentially targeted a cellular component endoplasmic reticulum (ER) and in small nucleolar RNA fragments (snoRFs), the molecular function protein binding. To summarize, our work demonstrated that the progeny of heat-stressed plants exhibit changes in the expression of tRFs and snoRFs but not of small nuclear RNA fragments (snRFs) or ribosomal RNA fragments (rRFs) and these changes likely better prepare the progeny of stressed plants to future stress encounters.