Genome-wide identification and in-silico expression analysis of CCO gene family in sunflower (Helianthus annnus) against abiotic stress.

Carotenoid cleavage oxygenases (CCOs) enzymes play an important role in plant growth and development by producing a wide array of apocarotenoids and their derivatives. These compounds are vital for colouring flowers and fruits and synthesizing plant hormones such as abscisic acid and strigolactones. Despite their importance, the gene family responsible for CCO enzymes in sunflowers has not been identified. In this study, we identify the CCO genes of the sunflower plant to fill this knowledge gap. Phylogenetic and synteny analysis indicated that the Helianthus annnus CCO (HaCCO) genes were conserved in different plant species and they could be divided into three subgroups based on their conserved domains. Analysis using MEME tool and multiple sequence alignment identified conserved motifs in the HaCCO gene sequence. Cis-regulatory elements (CREs) analysis of the HaCCO genes indicated the presence of various responsive elements related to plant hormones, development, and responses to both biotic and abiotic stresses. This implies that these genes may respond to plant hormones, developmental cues, and drought stress, offering potential applications in the development of more resistant crops. Genes belonging to the 9-cis-epoxy carotenoid dioxygenases (NCED) subgroups predominantly exhibited chloroplast localization, whereas the genes found in other groups are primarily localized in the cytoplasm. These 21 identified HaCCOs were regulated by 60 miRNAs, indicating the crucial role of microRNAs in gene regulation in sunflowers. Gene expression analysis under drought stress revealed significant up-regulation of HaNCED16 and HaNCED19, genes that are pivotal in ABA hormone biosynthesis. During organ-specific gene expression analysis, HaCCD12 and HaCCD20 genes exhibit higher activity in leaves, indicating a potential role in leaf pigmentation. This study provides a foundation for future research on the regulation and functions of the CCO gene family in sunflower and beyond. There is potential for developing molecular markers that could be employed in breeding programs to create new sunflower lines resistant to biotic and abiotic stresses.

Genome-Wide Identification and Expression Analysis of TGA Family Genes Associated with Abiotic Stress in Sunflowers (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) is an important, substantial global oil crop with robust resilience to drought and salt stresses. The TGA (TGACG motif-binding factor) transcription factors, belonging to the basic region leucine zipper (bZIP) family, have been implicated in orchestrating multiple biological processes. Despite their functional significance, a comprehensive investigation of the TGA family's abiotic stress tolerance in sunflowers remains elusive. In the present study, we identified 14 TGA proteins in the sunflower genome, which were unequally distributed across 17 chromosomes. Employing phylogenetic analysis encompassing 149 TGA members among 13 distinct species, we revealed the evolutionary conservation of TGA proteins across the plant kingdom. Collinearity analysis suggested that both HaTGA01 and HaTGA03 were generated due to HaTGA08 gene duplication. Notably, qRT-PCR analysis demonstrated that HaTGA04, HaTGA05, and HaTGA14 genes were remarkably upregulated under ABA, MeJA, and salt treatments, whereas HaTGA03, HaTGA06, and HaTGA07 were significantly repressed. This study contributes valuable perspectives on the potential roles of the HaTGA gene family under various stress conditions in sunflowers, thereby enhancing our understanding of TGA gene family dynamics and function within this agriculturally significant species.

A cluster of putative resistance genes is associated with a dominant resistance to sunflower broomrape.

KEY MESSAGE: The HaOr5 resistance gene is located in a large genomic insertion containing putative resistance genes and provides resistance to O. cumana, preventing successful connection to the sunflower root vascular system. Orobanche cumana (sunflower broomrape) is a parasitic plant that is part of the Orobanchaceae family and specifically infests sunflower crops. This weed is an obligate parasitic plant that does not carry out photosynthetic activity or develop roots and is fully dependent on its host for its development. It produces thousands of dust-like seeds per plant. It possesses a high spreading ability and has been shown to quickly overcome resistance genes successively introduced by selection in cultivated sunflower varieties. The first part of its life cycle occurs underground. The connection to the sunflower vascular system is essential for parasitic plant survival and development. The HaOr5 gene provides resistance to sunflower broomrape race E by preventing the connection of O. cumana to the root vascular system. We mapped a single position of the HaOr5 gene by quantitative trait locus mapping using two segregating populations. The same location of the HaOr5 gene was identified by genome-wide association. Using a large population of thousands of F2 plants, we restricted the location of the HaOr5 gene to a genomic region of 193 kb. By sequencing the whole genome of the resistant line harboring the major resistance gene HaOr5, we identified a large insertion of a complex genomic region containing a cluster of putative resistance genes.

Genome-wide identification and characterization of protein phosphatase 2C (PP2C) gene family in sunflower (Helianthus annuus L.) and their expression profiles in response to multiple abiotic stresses.

Plant protein phosphatase 2C (PP2C) plays vital roles in responding to various stresses, stimulating growth factors, phytohormones, and metabolic activities in many important plant species. However, the PP2C gene family has not been investigated in the economically valuable plant species sunflower (Helianthus annuus L.). This study used comprehensive bioinformatics tools to identify and characterize the PP2C gene family members in the sunflower genome (H. annuus r1.2). Additionally, we analyzed the expression profiles of these genes using RNA-seq data under four different stress conditions in both leaf and root tissues. A total of 121 PP2C genes were identified in the sunflower genome distributed unevenly across the 17 chromosomes, all containing the Type-2C phosphatase domain. HanPP2C genes are divided into 15 subgroups (A-L) based on phylogenetic tree analysis. Analyses of conserved domains, gene structures, and motifs revealed higher structural and functional similarities within various subgroups. Gene duplication and collinearity analysis showed that among the 53 HanPP2C gene pairs, 48 demonstrated segmental duplications under strong purifying selection pressure, with only five gene pairs showing tandem duplications. The abundant segmental duplication was observed compared to tandem duplication, which was the major factor underlying the dispersion of the PP2C gene family in sunflowers. Most HanPP2C proteins were localized in the nucleus, cytoplasm, and chloroplast. Among the 121 HanPP2C genes, we identified 71 miRNAs targeting 86 HanPP2C genes involved in plant developmental processes and response to abiotic stresses. By analyzing cis-elements, we identified 63 cis-regulatory elements in the promoter regions of HanPP2C genes associated with light responsiveness, tissue-specificity, phytohormone, and stress responses. Based on RNA-seq data from two sunflower tissues (leaf and root), 47 HanPP2C genes exhibited varying expression levels in leaf tissue, while 49 HanPP2C genes showed differential expression patterns in root tissue across all stress conditions. Transcriptome profiling revealed that nine HanPP2C genes (HanPP2C12, HanPP2C36, HanPP2C38, HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73) exhibited higher expression in leaf tissue, and five HanPP2C genes (HanPP2C13, HanPP2C47, HanPP2C48, HanPP2C54, and HanPP2C95) showed enhanced expression in root tissue in response to the four stress treatments, compared to the control conditions. These results suggest that these HanPP2C genes may be potential candidates for conferring tolerance to multiple stresses and further detailed characterization to elucidate their functions. From these candidates, 3D structures were predicted for six HanPP2C proteins (HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73), which provided satisfactory models. Our findings provide valuable insights into the PP2C gene family in the sunflower genome, which could play a crucial role in responding to various stresses. This information can be exploited in sunflower breeding programs to develop improved cultivars with increased abiotic stress tolerance.

Application of machine learning for identification of heterotic groups in sunflower through combined approach of phenotyping, genotyping and protein profiling.

Application of machine learning in plant breeding is a recent concept, that has to be optimized for precise utilization in the breeding program of high yielding crop plants. Identification and efficient utilization of heterotic grouping pattern aided with machine learning approaches is of utmost importance in hybrid cultivar breeding as it can save time and resources required to breed a new plant hybrid/variety. In the present study, 109 genotypes of sunflower were investigated at morphological, biochemical (SDS-PAGE) and molecular levels (through micro-satellites (SSR) markers) for heterotic grouping. All the three datasets were combined, scaled, and subjected to unsupervised machine learning algorithms, i.e., Hierarchical clustering, K-means clustering and hybrid clustering algorithm (hierarchical + K-means) for assessment of efficiency and resolution power of these algorithms in practical plant breeding for heterotic grouping identification. Following the application of machine learning unsupervised clustering approach, two major groups were identified in the studied sunflower germplasm, and further classification revealed six smaller classes in each major group through hierarchical and hybrid clustering approach. Due to high resolution, obtained in hierarchical clustering, classification achieved through this algorithm was further used for selection of potential parents. One genotype from each smaller group was selected based on the maximum seed yield potential and hybridized in a line × tester mating design producing 36 F1 cross combinations. These F1s along with their parents were studied in open field conditions for validating the efficacy of identified heterotic groups in sunflowers genetic material under study. Data for 11 agronomic and qualitative traits were recorded. These 36 F1 combinations were tested for their combining ability (General/Specific), heterosis, genotypic and phenotypic correlation and path analysis. Results suggested that F1 hybrids performed better for all the traits under investigation than their respective parents. Findings of the study validated the use of machine learning approaches in practical plant breeding; however, more accurate and robust clustering algorithms need to be developed to handle the data noisiness of open field experiments.

Comparative transcriptome and coexpression network analysis reveals key pathways and hub candidate genes associated with sunflower (Helianthus annuus L.) drought tolerance.

BACKGROUND: Drought severely limits sunflower production especially at the seedling stage. To investigate the response mechanism of sunflowers to drought stress, we utilized two genotypes of sunflower materials with different drought resistances as test materials. The physiological responses were investigated under well-watered (0 h) and drought-stressed conditions (24 h, 48 h, and 72 h). RESULTS: ANOVA revealed the greatest differences in physiological indices between 72 h of drought stress and 0 h of drought stress. Transcriptome analysis was performed after 72 h of drought stress. At 0 h, there were 7482 and 5627 differentially expressed genes (DEGs) in the leaves of K55 and K58, respectively, and 2150 and 2527 DEGs in the roots of K55 and K58, respectively. A total of 870 transcription factors (TFs) were identified among theDEGs, among which the high-abundance TF families included AP2/ERF, MYB, bHLH,and WRKY. Five modules were screened using weighted gene coexpressionnetwork analysis (WGCNA), three and two of which were positively and negatively, respectively, related to physiological traits. KEGG analysis revealedthat under drought stress, "photosynthesis", "carotenoid biosynthesis", "starch and sucrose metabolism", "ribosome", "carotenoid biosynthesis", "starch and sucrose metabolism", "protein phosphorylation" and "phytohormone signaling" are six important metabolic pathways involved in the response of sunflower to drought stress. Cytoscape software was used to visualize the three key modules, and the hub genes were screened. Finally, a total of 99 important candidate genes that may be associated with the drought response in sunflower plants were obtained, and the homology of these genes was compared with that in Arabidopsis thaliana. CONCLUSIONS: Taken together, our findings could lead to a better understanding of drought tolerance in sunflowers and facilitate the selection of drought-tolerant sunflower varieties.

Genome-wide identification and expression analysis of the Dof gene family reveals their involvement in hormone response and abiotic stresses in sunflower (Helianthus annuus L.).

DNA binding with one finger (Dof), plant-specific zinc finger transcription factors, can participate in various physiological and biochemical processes during the life of plants. As one of the most important oil crops in the world, sunflower (Helianthus annuus L.) has significant economic and ornamental value. However, a systematic analysis of H. annuus Dof (HaDof) members and their functions has not been extensively conducted. In this study, we identified 50 HaDof genes that are unevenly distributed on 17 chromosomes of sunflower. We present a comprehensive overview of the HaDof genes, including their chromosome locations, phylogenetic analysis, and expression profile characterization. Phylogenetic analysis classified the 366 Dof members identified from 11 species into four groups (further subdivided into nine subfamilies). Segmental duplications are predominantly contributed to the expansion of sunflower Dof genes, and all segmental duplicate gene pairs are under purifying selection due to strong evolutionary constraints. Furthermore, we observed differential expression patterns for HaDof genes in normal tissues as well as under hormone treatment or abiotic stress conditions by analyzing RNA-seq data from previous studies and RT-qPCR data in our current study. The expression of HaDof04 and HaDof43 were not detected in any samples, which implied that they may be gradually undergoing pseudogenization process. Some HaDof genes, such as HaDof25 and HaDof30, showed responsiveness to exogenous plant hormones, such as kinetin, brassinosteroid, auxin or strigolactone, while others like HaDof15 and HaDof35 may participate in abiotic stress resistance of sunflower seedling. Our study represents the initial step towards understanding the phylogeny and expression characterization of sunflower Dof family genes, which may provide valuable reference information for functional studies on hormone response, abiotic stress resistance, and molecular breeding in sunflower and other species.

Characterization of PYL gene family and identification of HaPYL genes response to drought and salt stress in sunflower.

In the context of global climate change, drought and soil salinity are some of the most devastating abiotic stresses affecting agriculture today. PYL proteins are essential components of abscisic acid (ABA) signaling and play critical roles in responding to abiotic stressors, including drought and salt stress. Although PYL genes have been studied in many species, their roles in responding to abiotic stress are still unclear in the sunflower. In this study, 19 HaPYL genes, distributed on 15 of 17 chromosomes, were identified in the sunflower. Fragment duplication is the main cause of the expansion of PYL genes in the sunflower genome. Based on phylogenetic analysis, HaPYL genes were divided into three subfamilies. Members in the same subfamily share similar protein motifs and gene exon-intron structures, except for the second subfamily. Tissue expression patterns suggested that HaPYLs serve different functions when responding to developmental and environmental signals in the sunflower. Exogenous ABA treatment showed that most HaPYLs respond to an increase in the ABA level. Among these HaPYLs, HaPYL2a, HaPYL4d, HaPYL4g, HaPYL8a, HaPYL8b, HaPYL8c, HaPYL9b, and HaPYL9c were up-regulated with PEG6000 treatment and NaCl treatment. This indicates that they may play a role in resisting drought and salt stress in the sunflower by mediating ABA signaling. Our findings provide some clues to further explore the functions of PYL genes in the sunflower, especially with regards to drought and salt stress resistance.

Development and characterization of a new sunflower source of resistance to race G of Orobanche cumana Wallr. derived from Helianthus anomalus.

KEY MESSAGE: A new OrAnom1 gene introgressed in cultivated sunflower from wild Helianthus anomalus confers late post-attachment resistance to Orobanche cumana race G and maps to a target interval in Chromosome 4 where two receptor-like kinases (RLKs) have been identified in the H. anomalus genome as putative candidates. Sunflower broomrape is a parasitic weed that infects sunflower (Helianthus annuus L.) roots causing severe yield losses. Breeding for resistance is the most effective and sustainable control method. In this study, we report the identification, introgression, and genetic and physiological characterization of a new sunflower source of resistance to race G of broomrape developed from the wild annual sunflower H. anomalus (accession PI 468642). Crosses between PI 468642 and the susceptible line P21 were carried out, and the genetic study was conducted in BC1F1, BC1F2, and its derived BC1F3 populations. A BC1F5 germplasm named ANOM1 was developed through selection for race G resistance and resemblance to cultivated sunflower. The resistant trait showed monogenic and dominant inheritance. The gene, named OrAnom1, was mapped to Chromosome 4 within a 1.2 cM interval and co-segregated with 7 SNP markers. This interval corresponds to a 1.32 Mb region in the sunflower reference genome, housing a cluster of receptor-like kinase and receptor-like protein (RLK-RLP) genes. Notably, the analysis of the H. anomalus genome revealed the absence of RLPs in the OrAnom1 target region but featured two RLKs as possible OrAnom1 candidates. Rhizotron and histological studies showed that OrAnom1 determines a late post-attachment resistance mechanism. Broomrape can establish a vascular connection with the host, but parasite growth is stopped before tubercle development, showing phenolic compounds accumulation and tubercle necrosis. ANOM1 will contribute to broadening the genetic basis of broomrape resistance in the cultivated sunflower pool and to a better understanding of the molecular basis of the sunflower-broomrape interaction.

Genetic control of abiotic stress-related specialized metabolites in sunflower.

BACKGROUND: Abiotic stresses in plants include all the environmental conditions that significantly reduce yields, like drought and heat. One of the most significant effects they exert at the cellular level is the accumulation of reactive oxygen species, which cause extensive damage. Plants possess two mechanisms to counter these molecules, i.e. detoxifying enzymes and non-enzymatic antioxidants, which include many classes of specialized metabolites. Sunflower, the fourth global oilseed, is considered moderately drought resistant. Abiotic stress tolerance in this crop has been studied using many approaches, but the control of specialized metabolites in this context remains poorly understood. Here, we performed the first genome-wide association study using abiotic stress-related specialized metabolites as molecular phenotypes in sunflower. After analyzing leaf specialized metabolites of 450 hybrids using liquid chromatography-mass spectrometry, we selected a subset of these compounds based on their association with previously known abiotic stress-related quantitative trait loci. Eventually, we characterized these molecules and their associated genes. RESULTS: We putatively annotated 30 compounds which co-localized with abiotic stress-related quantitative trait loci and which were associated to seven most likely candidate genes. A large proportion of these compounds were potential antioxidants, which was in agreement with the role of specialized metabolites in abiotic stresses. The seven associated most likely candidate genes, instead, mainly belonged to cytochromes P450 and glycosyltransferases, two large superfamilies which catalyze greatly diverse reactions and create a wide variety of chemical modifications. This was consistent with the high plasticity of specialized metabolism in plants. CONCLUSIONS: This is the first characterization of the genetic control of abiotic stress-related specialized metabolites in sunflower. By providing hints concerning the importance of antioxidant molecules in this biological context, and by highlighting some of the potential molecular mechanisms underlying their biosynthesis, it could pave the way for novel applications in breeding. Although further analyses will be required to better understand this topic, studying how antioxidants contribute to the tolerance to abiotic stresses in sunflower appears as a promising area of research.

Integrated transcriptome and metabolome analysis revealed that HaMYB1 modulates anthocyanin accumulation to deepen sunflower flower color.

KEY MESSAGE: HanMYB1 was found to play positive roles in the modulation of anthocyanins metabolism based on the integrative analysis of different color cultivars and the related molecular genetic analyses. As a high value ornamental and edible crop with various colors, sunflowers (Helianthus annuus L.) provide an ideal system to understand the formation of flower color. Anthocyanins are major pigments in higher plants, which is associated with development of flower colors and ability of oxidation resistance. Here, we performed an integrative analysis of the transcriptome and flavonoid metabolome in five sunflower cultivars with different flower colors. According to differentially expressed genes and differentially accumulated flavonoids, these cultivars could be grouped into yellow and red. The results showed that more anthocyanins were accumulated in the red group flowers, especially the chrysanthemin. Some anthocyanins biosynthesis-related genes like UFGT (UDP-glycose flavonoid glycosyltransferase) also expressed more in the red group flowers. A MYB transcriptional factor, HanMYB1, was found to play vital positive roles in the modulation of anthocyanins metabolism by the integrative analysis. Overexpressed HanMYB1 in tobacco could deepen the flower color, increase the accumulation of anthocyanins and directly active the express of UFGT genes. Our findings indicated that the MYB transcriptional factors provide new insight into the dynamic regulation of the anthocyanin biosynthesis in facilitating sunflower color formation and anthocyanin accumulation.

The genomic basis of nitrogen utilization efficiency and trait plasticity to improve nutrient stress tolerance in cultivated sunflower.

Maintaining crop productivity is challenging as population growth, climate change, and increasing fertilizer costs necessitate expanding crop production to poorer lands whilst reducing inputs. Enhancing crops' nutrient use efficiency is thus an important goal, but requires a better understanding of related traits and their genetic basis. We investigated variation in low nutrient stress tolerance in a diverse panel of cultivated sunflower genotypes grown under high and low nutrient conditions, assessing relative growth rate (RGR) as performance. We assessed variation in traits related to nitrogen utilization efficiency (NUtE), mass allocation, and leaf elemental content. Across genotypes, nutrient limitation generally reduced RGR. Moreover, there was a negative correlation between vigor (RGR in control) and decline in RGR in response to stress. Given this trade-off, we focused on nutrient stress tolerance independent of vigor. This tolerance metric correlated with the change in NUtE, plasticity for a suite of morphological traits, and leaf element content. Genome-wide associations revealed regions associated with variation and plasticity in multiple traits, including two regions with seemingly additive effects on NUtE change. Our results demonstrate potential avenues for improving sunflower nutrient stress tolerance independent of vigor, and highlight specific traits and genomic regions that could play a role in enhancing tolerance.

The genomic consequences of selection across development.

Understanding how natural selection drives diversification in nature has been at the forefront of biological research for over a century. The main idea is simple: natural selection favours individuals best suited to pass on their genes. However, the journey from birth to reproduction is complex as organisms experience multiple developmental stages, each influenced by genetic and environmental factors (Orr, 2009). These complexities compound even further as each stage of development might be governed by a unique underlying set of alleles and genes. In this issue of Molecular Ecology, Goebl et al. (2022) examine the role of natural selection in driving ecotypic divergence across different life history stages of the prairie sunflower Helianthus petiolaris. The authors used reciprocal transplant experiments, demographic models, and genomic sequencing to explore fitness variation across developmental stages. They show how natural selection impacts population divergence across multiple life history stages and evaluate the resulting allele frequency changes. Goebl et al. link these results to the role of chromosomal inversions, thus furthering our understanding of how ecological divergence proceeds in the face of gene flow. Below, we explore these results in detail and complement their interpretation by considering the evolution of genetic correlations amongst traits governing fitness.

Shading treatment during late stage of seed development promotes subsequent seed germination and seedlings establishment in sunflower.

During the sunflower seed production process, the role of artificial shading treatment (ST) in seed development and subsequent seed germination remains largely unknown. In the present study, sunflower mother plants were artificially shaded during 1-34 (full period-ST, FST), 1-22 (early period-ST, EST), and 22-34 (late period-ST, LST) days after pollination (DAP), to examine the effects of parental shading on subsequent seed germination. Both FST and EST significantly reduced the photosynthetic efficiency of sunflower, manifested as decreased seed dry weight and unfavorable seed germination. On the contrary, LST remarkably increased seed dry weight and promoted subsequent seed germination and seedling establishment. LST enhanced the activities of several key enzymes involved in triglyceride anabolism and corresponding-genes expression, which in turn increased the total fatty acid contents and altered the fatty acid composition. During early germination, the key enzyme activities involved in triglyceride disintegration and corresponding-gene expressions in LST seeds were apparently higher than those in seeds without the shading treatment (WST). Consistently, LST seeds had significant higher contents of ATP and soluble sugar. Moreover, enzyme activities related to abscisic acid (ABA) biosynthesis and corresponding gene expressions decreased within LST seeds, whereas the enzyme activities and corresponding gene expressions associated with gibberellin (GA) biosynthesis were increased. These results were also evidenced by the reduced ABA content but elevated GA level within LST seeds, giving rise to higher GA/ABA ratio. Our findings suggested that LST could promote sunflower seed development and subsequent seed germination as well as seedling establishment through modulating the dynamic metabolism of triglycerides, fatty acid and GA/ABA balance.

HaMYBA-HabHLH1 regulatory complex and HaMYBF fine-tune red flower coloration in the corolla of sunflower (Helianthus annuus L.).

Sunflowers are well-known ornamental plants, while sunflowers with red corolla are rare and the mechanisms underlying red coloration remain unclear. Here, a comprehensive analysis of metabolomics and transcriptomics on flavonoid pathway was performed to investigate the molecular mechanisms underlying the differential color formation between red sunflower Pc103 and two yellow sunflowers (Yr17 and Y35). Targeted metabolomic analysis revealed higher anthocyanin levels but lower flavonol content in Pc103 compared to the yellow cultivars. RNA-sequencing and phylogenetic analysis identified multiple genes involved in the flavonoid pathway, including series of structural genes and three MYB and bHLH genes. Specifically, HaMYBA and HabHLH1 were up-regulated in Pc103, whereas HaMYBF exhibited reduced expression. HaMYBA was found to interact with HabHLH1 in vivo and in vitro, while HaMYBF does not. Transient expression analysis further revealed that HabHLH1 and HaMYBA cooperatively regulate increased expression of dihydroflavonol 4-reductase (DFR), leading to anthocyanin accumulation. On the other hand, ectopic expression of HaMYBF independently modulates flavonol synthase (FLS) expression, but hindered anthocyanin production. Collectively, our findings suggest that the up-regulation of HaMYBA and HabHLH1, as well as the down-regulation of HaMYBF, contribute to the red coloration in Pc103. It offers a theoretical basis for improving sunflower color through genetic engineering.

Identification and expression analysis of miR396 and its target genes in Jerusalem artichoke under temperature stress.

The highly conserved miR396 plays a pivotal role in the growth, development, and responses to abiotic and biotic stresses in plants. However, research on miR396 and its targets in Jerusalem artichoke remains largely unexplored. In this study, we employed bioinformatics and experimental techniques, such as cloning and qRT-PCR, to investigate the regulatory role of miR396 on its targets, leveraging our lab's transcriptomic and degradomic data of Jerusalem artichoke. Specifically, we initially cloned and characterized the precursors (htu-MIR396a/b/c) and mature sequences (htu-miR396a/b/c) of three miR396 isoforms. Subsequently, we identified nine target genes, including seven Growth-Regulating Factors (GRFs) (HtGRF3/4/6/9/10/12/13), one WRKY transcription factor (HtWRKY40), and one Scarecrow-like (SCL) transcription factor (HtSCL33). Finally, we conducted an analysis of their expression patterns across various tissues and their responses to temperature stress. Notably, htu-MIR396s exhibited high expression in seedling stems, while htu-miR396s predominantly expressed in seedling leaves. Moreover, HtWRKY40 and HtSCL33 displayed higher expression levels than HtGRFs in most tissues, except leaves. Remarkably, HtGRF4/6/10/12/13 exhibited higher expression in leaves than in roots and stems during seedling growth. Furthermore, during tuber development, HtGRF4/6/10, HtWRKY40, and HtSCL33 were highly expressed, while HtGRF3/9/12/13 showed relatively lower expression levels. Under heat stress (42℃), htu-MIR396 expression was up-regulated, and htu-miR396 showed dynamic expression patterns in seedlings, resulting in the induction of HtGRF4/6/10/12/13 in leaves and HtSCL33 in roots, while HtWRKY40 in leaves was repressed. Conversely, under cold stress (4℃), htu-MIR396s showed fluctuating expression levels, and htu-miR396s were up-regulated in seedlings. Notably, HtGRF4/13 and HtSCL33 in seedlings were reduced, whereas HtGRF6 in roots and HtWRKY40 in leaves were enhanced. These findings offer valuable insights into the functional roles of miR396-target interactions under abiotic stress in Jerusalem artichoke.

Unveiling HSP40/60/70/90/100 gene families and abiotic stress response in Jerusalem artichoke.

Heat shock proteins (HSPs) are essential for plant growth, development, and stress adaptation. However, their roles in Jerusalem artichoke are largely unexplored. Using bioinformatics, we classified 143 HSP genes into distinct families: HSP40 (82 genes), HSP60 (22 genes), HSP70 (29 genes), HSP90 (6 genes), and HSP100 (4 genes). Our analysis covered their traits, evolution, and structures. Using RNA-seq data, we uncovered unique expression patterns of these HSP genes across growth stages and tissues. Notably, HSP40, HSP60, HSP70, HSP90, and HSP100 families each had specific roles. We also studied how these gene families responded to various stresses, from extreme temperatures to drought and salinity, revealing intricate expression dynamics. Remarkably, HSP40 showed remarkable flexibility, while HSP60, HSP70, HSP90, and HSP100 responded specifically to stress types. Moreover, our analysis unveiled significant correlations between gene pairs under stress, implying cooperative interactions. qRT-PCR validation underscored the significance of particular genes such as HtHSP60-7, HtHSP90-5, HtHSP100-2, and HtHSP100-3 in responding to stress. In summary, our study advances the understanding of how HSP gene families collectively manage stresses in Jerusalem artichoke. This provides insights into specific gene functions and broader plant stress responses.

Repeatability of adaptation in sunflowers reveals that genomic regions harbouring inversions also drive adaptation in species lacking an inversion.

Local adaptation commonly involves alleles of large effect, which experience fitness advantages when in positive linkage disequilibrium (LD). Because segregating inversions suppress recombination and facilitate the maintenance of LD between locally adapted loci, they are also commonly found to be associated with adaptive divergence. However, it is unclear what fraction of an adaptive response can be attributed to inversions and alleles of large effect, and whether the loci within an inversion could still drive adaptation in the absence of its recombination-suppressing effect. Here, we use genome-wide association studies to explore patterns of local adaptation in three species of sunflower: Helianthus annuus, Helianthus argophyllus, and Helianthus petiolaris, which each harbour a large number of species-specific inversions. We find evidence of significant genome-wide repeatability in signatures of association to phenotypes and environments, which are particularly enriched within regions of the genome harbouring an inversion in one species. This shows that while inversions may facilitate local adaptation, at least some of the loci can still harbour mutations that make substantial contributions without the benefit of recombination suppression in species lacking a segregating inversion. While a large number of genomic regions show evidence of repeated adaptation, most of the strongest signatures of association still tend to be species-specific, indicating substantial genotypic redundancy for local adaptation in these species.

In plants, like in humans, DNA is arranged into sections known as genes that are in turn organised into structures called chromosomes. Mutations that modify the activity of these genes can help plant species to adapt to a new environment or to extreme conditions such as drought. However, successful adaptation often requires changes in many different genes. If these sets of genes are located close to each other on the same chromosome, any mutations will likely be passed onto the next generation together. If the genes are located further away, or even on different chromosomes, they may instead be inherited separately so that the next generation does not benefit as much from the adaptation. A chromosome inversion ­ when a segment of chromosome breaks off and reattaches the other way around ­ can increase the likelihood that sets of mutations on the same chromosome will be inherited together. Many previous studies have found that chromosome inversions tend to drive the ability of species to adapt to different environments by keeping together mutations that affect the same characteristics. However, it is not clear how inversions affect the repeatability of the adaptation, that is, if another group of closely related plants faced the same challenge in their environment would they evolve in the same way, or would they evolve a new response? To address this question, Soudi, Jahani et al. used a genetics approach known as a genome wide association study to explore how three closely related species of sunflower have adapted to their respective environments. Two of the species grow in various environments across the centre and west of the USA that are often hot and dry, whereas the third species is restricted to the more humid coastal plain of Texas, USA. The experiments found that a few key genes had changed in all three sunflower species. However, each species also had mutations in a larger set of unique genes that were not changed in the other species. Regions of chromosomes harbouring inversions in one of the species tended to have more of the key genes within them, compared to other genomic regions. This was also true for species that did not have inversions in those regions. This demonstrates that genes in regions affected by chromosome inversions can still help plants adapt to changes in the environment even in the absence of inversions. Sunflowers are widely grown for their edible oily seeds. In the future, some of the key genes identified in this work may be useful candidates for plant breeding to improve the resilience of sunflowers to drought, high temperatures and other environmental challenges.

VTE1 and VTE3 Gene Expression During Vitamin E Production in Sunflower (Helianthus annuus L.) Treated with Different Fertilization.

<b>Background and Objective:</b> Sunflower is one of the important commodities in agriculture. The oil content in sunflower seeds has been widely used as cooking oil, but in Indonesia, the utilization of this oil is still relatively low. In addition, sunflowers also contain vitamin E which is useful as an antioxidant, so it can be used to reduce the risk of cardiovascular disease. This study aims to determine gene expression at the RNA level towards vitamin E biosynthesis using different fertilization treatments. <b>Materials and Methods:</b> Sunflowers that had been given different fertilizers were taken in three flowering phases, R3, R5 and R8. Flower samples were isolated until RNA was obtained. The isolation results were tested using real-time PCR to determine the relative gene expression of the <i>VTE1</i> and <i>VTE3</i> genes. After the sunflower seeds were fully ripe, vitamin E content was tested in each treatment and the results were compared with the relative gene expression obtained. <b>Results:</b> The results obtained were fluctuating, but in general, the relative gene expression obtained in the <i>VTE1</i> gene increased in the R3 phase and then decreased in the R5 and R8 phases. Whereas, in the <i>VTE3</i> gene, the relative gene expression obtained experienced an increase in the R3 and R5 phases and then decreased in the R8 phase. The highest vitamin E content was obtained by sample P3 (4218 µg mL¹) and the lowest was obtained by sample P2 (1798 µg mL¹). <b>Conclusion:</b> A balanced ratio of 92:46:30 kg ha¹ of major nutrient fertilizer involving N, P and K could increase vitamin E content in sunflowers. Such a combination exhibited stable expression of the <i>VTE1</i> and <i>VTE3</i> genes in all phases of flowering.

Comparative genomics of light harvesting chlorophyll (LHC) gene family and impact of chlorophyll-A contents under drought stress in Helianthus annuus.

Drought is one of the main environmental stressors that can alter the water status of plants; negatively affect growth, assimilation, and photosynthesis; and eventually reduce crop yield. We explored the dependence of drought tolerance traits on chlorophyll-A content. Local sunflower cultivars (FH-01, FH-628, FH-633, FH-572, and FH-653) were grown in pots and subjected to drought by withholding water for 10, 15, or 20 d. One month after germination, the leaves of the treated and non-treated plants were collected and subjected to biochemical analyses. Under different water stress levels, the levels of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and proline increased, whereas those of chlorophyll-A decreased. Regression analysis clearly found that proline (-0.442), POD (-0.528), SOD (-0.532), and CAT (-0.814) have negative beta coefficient values. Phylogenetic analysis revealed that the LHC gene family is divided into six clades. Subcellular locations indicated that most LHC genes were located in the chloroplast; however, only few genes were present in the peroxisomes and endoplasmic reticulum. Our research found that Arabidopsis thaliana LHC genes were highly homologous to the LHC genes of Helianthus annuus. Furthermore, the LHC genes of both species are located in the chloroplasts; therefore, they play a role in photosynthesis and renewable energy production. This study opens a new horizon for discussing the role of chlorophyll-A in the drought-related traits of sunflowers.