Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

microRNAs as Master Regulators of the Plant NB-LRR Defense Gene Family via the Production of Phased, Trans-acting siRNAs

The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (eg. Nmcas9, Sacas9, Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.

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CRISPR for Crop Improvement: An Update Review.

The microRNAs (miRNAs) are small (20-24 nt) sized, non-coding, single stranded riboregulator RNAs abundant in higher organisms. Recent findings have established that plants assign miRNAs as critical post-transcriptional regulators of gene expression in sequence-specific manner to respond to numerous abiotic stresses they face during their growth cycle. These small RNAs regulate gene expression via translational inhibition. Usually, stress induced miRNAs downregulate their target mRNAs, whereas, their downregulation leads to accumulation and function of positive regulators. In the past decade, investigations were mainly aimed to identify plant miRNAs, responsive to individual or multiple environmental factors, profiling their expression patterns and recognizing their roles in stress responses and tolerance. Altered expressions of miRNAs implicated in plant growth and development have been reported in several plant species subjected to abiotic stress conditions such as drought, salinity, extreme temperatures, nutrient deprivation, and heavy metals. These findings indicate that miRNAs may hold the key as potential targets for genetic manipulations to engineer abiotic stress tolerance in crop plants. This review is aimed to provide recent updates on plant miRNAs, their biogenesis and functions, target prediction and identification, computational tools and databases available for plant miRNAs, and their roles in abiotic stress-responses and adaptive mechanisms in major crop plants. Besides, the recent case studies for overexpressing the selected miRNAs for miRNA-mediated enhanced abiotic stress tolerance of transgenic plants have been discussed.

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MicroRNAs As Potential Targets for Abiotic Stress Tolerance in Plants

Increasing temperature and consequent changes in climate adversely affect plant growth and development, resulting in catastrophic loss of wheat productivity. For each degree rise in temperature, wheat production is estimated to reduce by 6%. A detailed overview of morpho-physiological responses of wheat to heat stress may help formulating appropriate strategies for heat-stressed wheat yield improvement. Additionally, searching for possible management strategies may increase productivity and sustainability of growing wheat. The major findings from this review are as follows: (1) heat stress significantly reduces seed germination and seedling growth, cell turgidity, and plant water-use efficiency; (2) at a cellular level, heat stress disturbs cellular functions through generating excessive reactive oxygen species, leading to oxidative stress; (3) the major responses of wheat to heat stress include the enhancement of leaf senescence, reduction of photosynthesis, deactivation of photosynthetic enzymes, and generation of oxidative damages to the chloroplasts; (4) heat stress also reduces grain number and size by affecting grain setting, assimilate translocation and duration and growth rate of grains; (5) effective approaches for managing heat stress in wheat include screening available germplasm under field trials and/or employing marker-assisted selection, application of exogenous protectants to seeds or plants, mapping quantitative trait locus conferring heat resistance and breeding; (6) a well-integrated genetic and agronomic management option may enhance wheat tolerance to heat. However, the success of applying various techniques of heat stress management requires greater understanding of heat tolerance features, molecular cloning, and characterization of genes. The overall success of the complex plant heat stress management depends on the concerted efforts of crop modelers, molecular biologists, and plant physiologists.

/pdf/heat-stress-effects-and-management-in-wheat-a-review-ap23v8zq1r.pdf

Heat stress effects and management in wheat. A review

Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression.

/pdf/the-bhlh-transcription-factor-bhlh104-interacts-with-iaa-99cg86yvs6.pdf

The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis

The human population is increasing at an alarming rate, whereas heavy metals (HMs) pollution is mounting serious environmental problem, which could lead to serious concern about the future sufficiency of global food production. Some HMs such as Mn, Cu, and Fe, at lower concentration serves as an essential vital component of plant cell as they are crucial in various enzyme catalyzed biochemical reactions. At higher concentration, a vast variety of HMs such as Mn, Cu, Cd, Fe, Hg, Al and As, impose toxic reaction in the plant system which greatly affect the crop yield. Recently, microRNAs (miRNAs) that are small class of non-coding riboregulator have emerged as central regulator of numerous abiotic stresses including HMs. Increasing reports indicate that plants have evolved specialized inbuilt mechanism viz. signal transduction, translocation and sequestration to counteract the toxic response of HMs. Combining computational and wet laboratory approaches have produced sufficient evidences concerning active involvement of miRNAs during HMs toxicity response by regulating various transcription factors and protein coding genes involved in plant growth and development. However, the direct role of miRNA in controlling various signaling molecules, transporters and chelating agents of HM metabolism is poorly understood. This review focuses on the latest progress made in the area of direct involvement of miRNAs in signaling, translocation and sequestration as well as recently added miRNAs in response to different HMs in plants.

MicroRNA mediated regulation of metal toxicity in plants: present status and future perspectives

MicroRNAs (miRNAs) are tiny non-coding regulatory molecules that modulate plant's gene expression either by cleaving or repressing their mRNA targets. To unravel the plant actions in response to various environmental factors, identification of stress related miRNAs is essential. For understanding the regulatory behaviour of various abiotic stresses and miRNAs in wheat genotype C-306, we examined expression profile of selected conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029 tangled in adapting osmotic, salt and cold stresses. The investigation revealed that two miRNAs (miR168, miR397) were down-regulated and miR172 was up-regulated under all the stress conditions. However, miR164 and miR1029 were up-regulated under cold and osmotic stresses in contrast to salt stress. miR529 responded to cold alone and does not change under osmotic and salt stress. miR393 showed up-regulation under osmotic and salt, and down-regulation under cold stress indicating auxin based differential cold response. Variation in expression level of studied miRNAs in presence of target genes delivers a likely elucidation of miRNAs based abiotic stress regulation. In addition, we reported new stress induced miRNAs Ta-miR855 using computational approach. Results revealed first documentation that miR855 is regulated by salinity stress in wheat. These findings indicate that diverse miRNAs were responsive to osmotic, salt and cold stress and could function in wheat response to abiotic stresses.

Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat

Plant’s secondary metabolites such as flavonoids, terpenoids and alkaloids etc. are known for their role in the defence against various insects-pests of plants and for medicinal benefits in human. Due to the immense biological importance of these phytochemicals, understanding the regulation of their biosynthetic pathway is crucial. In the recent past, advancement in the molecular technologies has enabled us to better understand the proteins, enzymes, genes, etc. involved in the biosynthetic pathway of the secondary metabolites. miRNAs are magical, tiny, noncoding ribonucleotides that function as critical regulators of gene expression in eukaryotes. Despite the accumulated knowledge of the miRNA-mediated regulation of several processes, the involvement of miRNAs in regulating secondary plant product biosynthesis is still poorly understood. Here, we summarize the recent progress made in the area of identification and characterizations of miRNAs involved in regulating the biosynthesis of secondary metabolites in plants and discuss the future perspectives for designing the viable strategies for their targeted manipulation.

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Contemporary Understanding of miRNA-Based Regulation of Secondary Metabolites Biosynthesis in Plants

Plants have evolved diverse mechanism to recognize pathogen attack and triggers defense responses. These defense responses alter host cellular function regulated by endogenous, small, non-coding miRNAs. To understand the mechanism of miRNAs regulated cellular functions during stem rust infection in wheat, we investigated eight different miRNAs viz. miR159, miR164, miR167, miR171, miR444, miR408, miR1129 and miR1138, involved in three different independent cellular defense response to infection. The investigation reveals that at the initiation of disease, accumulation of miRNAs might be playing a key role in hypersensitive response (HR) from host, which diminishes at the maturation stage. This suggests a possible host-fungal synergistic relation leading to susceptibility. Differential expression of these miRNAs in presence and absence of R gene provides a probable explanation of miRNA regulated R gene mediated independent pathways.

MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection.

Horticultural crops are an important part of agriculture for food as well as nutritional security. However, several pests and diseases along with adverse abiotic environmental factors poses a severe threat to these crops by affecting their quality and productivity. This warrants the effective and accelerated breeding programmes by utilizing innovative biotechnological tools that can tackle aforementioned issues. The recent technique of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) has greatly advanced the breeding for crop improvement due to its simplicity and high efficiency over other nucleases such as Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases. CRISPR/Cas9 tool contains a non-specific Cas9 nuclease and a single guide RNA that directs Cas9 to the specific genomic location creating double strand breaks and subsequent repair process creates insertion or deletion mutations. This is currently the widely adopted tool for reverse genetics, and crop improvement in large number of agricultural crops. The use of CRISPR/Cas9 in horticultural crops is limited to few crops due to lack of availability of regeneration protocols and sufficient sequence information in many horticultural crops. In this review, the present status of applicability of CRISPR/Cas9 in horticultural crops was discussed along with the challenges and future potential for possible improvement of these crops for their yield, quality and resistance to biotic and abiotic stress

/pdf/crispr-cas9-mediated-genome-engineering-for-improvement-of-1ucy0ms5ka.pdf

Om Prakash Gupta

Papers

MicroRNA mediated regulation of metal toxicity in plants: present status and future perspectives

Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat

Contemporary Understanding of miRNA-Based Regulation of Secondary Metabolites Biosynthesis in Plants

MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection.

CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops.