Identification of MYB gene family in medicinal tea tree Melaleuca alternifolia (Maiden and Betche) cheel and analysis of members regulating terpene biosynthesis.

BACKGROUND: The tea tree (Melaleuca alternifolia) is renowned for its production of tea tree oil, an essential oil primarily composed of terpenes extracted from its shoot. MYB transcription factors, which are one of the largest TF families, play a crucial role in regulating primary and secondary metabolite synthesis. However, knowledge of the MYB gene family in M. alternifolia is limited. METHODS AND RESULTS: Here, we conducted a comprehensive genome-wide analysis of MYB genes in M. alternifolia, referred to as MaMYBs, including phylogenetic relationships, structures, promoter regions, and GO annotations. Our findings classified 219 MaMYBs into four subfamilies: one 5R-MYB, four 3R-MYBs, sixty-one MYB-related, and the remaining 153 are all 2R-MYBs. Seven genes (MYB189, MYB146, MYB44, MYB29, MYB175, MYB162, and MYB160) were linked to terpenoid synthesis based on GO annotation. Phylogenetic analysis with Arabidopsis homologous MYB genes suggested that MYB193 and MYB163 may also be involved in terpenoid synthesis. Additionally, through correlation analysis of gene expression and metabolite content, we identified 42 MYB genes associated with metabolite content. CONCLUSION: The results provide valuable insights into the importance of MYB transcription factors in essential oil production in M. alternifolia. These findings lay the groundwork for a better understanding of the MYB regulatory network and the development of novel strategies to enhance essential oil synthesis in M. alternifolia.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit.

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.

Eco-friendly managements and molecular mechanisms for improving postharvest quality and extending shelf life of kiwifruit: A review.

Kiwifruit (Actinidia spp.) is a commercially important horticultural fruit crop worldwide. Kiwifruit contains numerous minerals, vitamins, and dietary phytochemicals, that not only responsible for the flavor but can also serve as adjuncts in the treatment of diabetes, digestive disorders, cardiovascular system, cancer and heart disease. However, fruit quality and shelf life affect consumer's acceptance and production chain. Understanding the methods of fruit storage preservation, as well as their biochemical, physiological, and molecular basis is essential. In recent years, eco-friendly (comprehensive and environmentally friendly) treatments such as hot water, ozone, chitosan, quercetin, and antifungal additive from biocontrol bacteria or yeast have been applied to improve postharvest fruit quality with longer shelf life. This review provides a comprehensive overview of the latest advancements in control measures, applications, and mechanisms related to water loss, chilling injury, and pathogen diseases in postharvest kiwifruit. Further studies should utilize genome editing techniques to enhance postharvest fruit quality and disease resistance through site-directed bio-manipulation of the kiwifruit genome.

Tomato sucrose transporter SlSUT4 participates in flowering regulation by modulating gibberellin biosynthesis.

The functions of sucrose transporters (SUTs) differ among family members. The physiological function of SUT1 has been studied intensively, while that of SUT4 in various plant species including tomato (Solanum lycopersicum) is less well-understood. In this study, we characterized the function of tomato SlSUT4 in the regulation of flowering using a combination of molecular and physiological analyses. SlSUT4 displayed transport activity for sucrose when expressed in yeast (Saccharomyces cerevisiae), and it localized at both the plasma membrane and tonoplast. SlSUT4 interacted with SlSUT1, causing partial internalization of the latter, the main phloem loader of sucrose in tomato. Silencing of SlSUT4 promoted SlSUT1 localization to the plasma membrane, contributing to increased sucrose export and thus increased sucrose level in the shoot apex, which promoted flowering. Both silencing of SlSUT4 and spraying with sucrose suppressed gibberellin biosynthesis through repression of ent-kaurene oxidase and gibberellin 20-oxidase-1 (2 genes encoding key enzymes in gibberellin biosynthesis) expression by SlMYB76, which directly bound to their promoters. Silencing of SlMYB76 promoted gibberellin biosynthesis. Our results suggest that SlSUT4 is a functional SUT in tomato; downregulation of SlSUT4 expression enhances sucrose transport to the shoot apex, which promotes flowering by inhibiting gibberellin biosynthesis.

Application of developmental regulators to improve in planta or in vitro transformation in plants.

Plant genetic transformation is a crucial step for applying biotechnology such as genome editing to basic and applied plant science research. Its success primarily relies on the efficiency of gene delivery into plant cells and the ability to regenerate transgenic plants. In this study, we have examined the effect of several developmental regulators (DRs), including PLETHORA (PLT5), WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), ENHANCED SHOOT REGENERATION (ESR1), WUSHEL (WUS) and a fusion of WUS and BABY-BOOM (WUS-P2A-BBM), on in planta transformation through injection of Agrobacterium tumefaciens in snapdragons (Antirrhinum majus). The results showed that PLT5, WIND1 and WUS promoted in planta transformation of snapdragons. An additional test of these three DRs on tomato (Solanum lycopersicum) further demonstrated that the highest in planta transformation efficiency was observed from PLT5. PLT5 promoted calli formation and regeneration of transformed shoots at the wound positions of aerial stems, and the transgene was stably inherited to the next generation in snapdragons. Additionally, PLT5 significantly improved the shoot regeneration and transformation in two Brassica cabbage varieties (Brassica rapa) and promoted the formation of transgenic calli and somatic embryos in sweet pepper (Capsicum annum) through in vitro tissue culture. Despite some morphological alternations, viable seeds were produced from the transgenic Bok choy and snapdragons. Our results have demonstrated that manipulation of PLT5 could be an effective approach for improving in planta and in vitro transformation efficiency, and such a transformation system could be used to facilitate the application of genome editing or other plant biotechnology application in modern agriculture.

The SUMO E3 Ligase MdSIZ1 Sumoylates a Cell Number Regulator MdCNR8 to Control Organ Size.

Plant growth and organ size putatively associated with crop yield are regulated by a complex network of genes including ones for controlling cell proliferation. The gene fw2.2 was first identified in tomatoes and reported to govern fruit size variation through controlling cell division. In this study, we isolated a putative ortholog of the tomato fw2.2 gene from apple, Cell Number Regulator 8 (MdCNR8). Our functional analysis showed that MdCNR8 may control fruit size and root growth. MdCNR8 was mediated by the SUMO E3 ligase MdSIZ1, and SUMOylation of MdCNR8 at residue-Lys39 promoted the translocation of MdCNR8 from plasma membrane to the nucleus. The effect of MdCNR8 in inhibiting root elongation could be completely counteracted by the coexpression of MdSIZ1. Moreover, the lower cell proliferation of apple calli due to silencing MdSIZ1 could be rescued by silencing MdCNR8. Collectively, our results showed that the MdSIZ1-mediated SUMOylation is required for the fulfillment of MdCNR8 in regulating cell proliferation to control plant organ size. This regulatory interaction between MdSIZ1 and MdCNR8 will facilitate understanding the mechanism underlying the regulation of organ size.

Genetic mapping reveals complex architecture and candidate genes involved in common bean response to Meloidogyne incognita infection.

Root-knot nematodes (RKNs), particularly Meloidogyne incognita, are among the most damaging and prevalent agricultural pathogens due to their ability to infect roots of almost all crops. The best strategy for their control is through the use of resistant cultivars. However, laborious phenotyping procedures make it difficult to assess nematode resistance in breeding programs. For common bean, this task is especially challenging because little has been done to discover resistance genes or markers to assist selection. We performed genome-wide association studies and quantitative trait loci mapping to explore the genetic architecture and genomic regions underlying the resistance to M. incognita and to identify candidate resistance genes. Phenotypic data were collected by a high-throughput assay, and the number of egg masses and the root-galling index were evaluated. Complex genetic architecture and independent genomic regions were associated with each trait. Single nucleotide polymorphisms on chromosomes Pv06, Pv07, Pv08, and Pv11 were associated with the number of egg masses, and SNPs on Pv01, Pv02, Pv05, and Pv10 were associated with root-galling. A total of 216 candidate genes were identified, including 14 resistance gene analogs and five differentially expressed in a previous RNA sequencing analysis. Histochemical analysis indicated that reactive oxygen species might play a role in the resistance response. Our findings open new perspectives to improve selection efficiency for RKN resistance, and the candidate genes are valuable targets for functional investigation and gene editing approaches.

EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in Arabidopsis.

The chloroplast is the main organelle for stress-induced production of reactive oxygen species (ROS). However, how chloroplastic ROS homeostasis is maintained under salt stress is largely unknown. We show that EGY3, a gene encoding a chloroplast-localized protein, is induced by salt and oxidative stresses. The loss of EGY3 function causes stress hypersensitivity while EGY3 overexpression increases the tolerance to both salt and chloroplastic oxidative stresses. EGY3 interacts with chloroplastic Cu/Zn-SOD2 (CSD2) and promotes CSD2 stability under stress conditions. In egy3-1 mutant plants, the stress-induced CSD2 degradation limits H2O2 production in chloroplasts and impairs H2O2-mediated retrograde signaling, as indicated by the decreased expression of retrograde-signal-responsive genes required for stress tolerance. Both exogenous application of H2O2 (or APX inhibitor) and CSD2 overexpression can rescue the salt-stress hypersensitivity of egy3-1 mutants. Our findings reveal that EGY3 enhances the tolerance to salt stress by promoting the CSD2 stability and H2O2-mediated chloroplastic retrograde signaling.

Effects of Maternal Environment on Seed Germination and Seedling Vigor of Petunia × hybrida under Different Abiotic Stresses.

Seed germination and seedling vigor can be affected by environmental cues experienced by the mother plant. However, information about how the maternal environment affects seed quality is scarce in ornamental plants. This study aimed to investigate the effects of two different maternal environments on the seed germination and seedling vigor of Petunia × hybrida under a variety of abiotic stresses. Petunia mother plants were grown in either a greenhouse during the summer months or an indoor controlled-temperature-and-light environment. Collected seeds were subjected to external stressors, including polyethylene glycol (PEG), sodium chloride (NaCl), high temperature, and abscisic acid (ABA), to determine seed germination percentage and seedling vigor. Results indicated that seeds harvested from the mother plants grown in a controlled environment germinated better than seeds harvested from the mother plants grown in the greenhouse when suboptimal germination conditions were applied. Additionally, the seedlings from the controlled maternal environment performed better in both ABA and salinity stress tests than the greenhouse seedlings. Interestingly, the greenhouse seedlings displayed less reactive oxygen species (ROS) damage and lower electrolyte leakage than the controlled environment seedlings under dehydration stress. The difference in germination and seedling vigor of seeds from the two different maternal environments might be due to the epigenetic memory inherited from the mother plants. This study highlighted the strong impact of the maternal environment on seed germination and seedling vigor in Petunia and may assist in high-quality seed production in ornamental plants.

Photosynthetic Responses of Anthurium × 'Red' under Different Light Conditions.

Light is an essential energy source for plant photosynthesis, although it can also be a stress-causing element. Therefore, the current research was aimed to compare photosynthetic responses of Anthurium × 'Red' leaves at different positions (bottom old leaf, 1; center mature leaf, 2; top expanded leaf, 3) established under three photosynthetic photon flux densities (PPFDs): 550 µmol·m-2·s-1 as high (H), 350 µmol·m-2·s-1 as medium (M), and 255 µmol·m-2·s-1 as low (L). After six months, all the replicates were relocated to interior rooms with a PPFD of 30 µmol·m-2·s-1. There were no significant differences in chlorophyll concentration of the old leaf among treatments, before (Day 0) and after shifting the plants to interior rooms (Day 30). The total chlorophyll concentrations of the mature and top leaves increased significantly. In greenhouse conditions, H and M treatments did not show any significant change for net photosynthetic rate (Pn) at various leaf positions. However, M2 exhibited an improved Pn in the interior conditions. Plants grown under M treatment were greener and had bigger leaves compared to other treatments. Our study reveals that Anthurium × 'Red' photosynthesis responses to different light conditions varied distinctly. However, M treatment can keep the plants looking green by accumulating enough energy for indoor conditions, and middle and lower leaves may be triggered to restore photosynthetic activity under low light or indoor conditions.

Interaction of BTB-TAZ protein MdBT2 and DELLA protein MdRGL3a regulates nitrate-mediated plant growth.

Nitrate acts as a vital signal molecule in the modulation of plant growth and development. The phytohormones gibberellin (GA) is also involved in this process. However, the exact molecular mechanism of how nitrate and GA signaling pathway work together in regulating plant growth remains poorly understood. In this study, we found that a nitrate-responsive BTB/TAZ protein MdBT2 participates in regulating nitrate-induced plant growth in apple (Malus × domestica). Yeast two-hybridization, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with a DELLA protein MdRGL3a, which is required for the ubiquitination and degradation of MdRGL3a proteins via a 26S proteasome-dependent pathway. Furthermore, heterologous expression of MdBT2 partially rescued growth inhibition caused by overexpression of MdRGL3a in Arabidopsis. Taken together, our findings indicate that MdBT2 promotes nitrate-induced plant growth partially through reducing the abundance of the DELLA protein MdRGL3a.

Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana.

BACKGROUND: To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. RESULTS: Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. CONCLUSIONS: The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

Genome-Wide Identification of the ABA Receptors Genes and Their Response to Abiotic Stress in Apple.

The pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) (known as PYLs for short) have been identified and characterized as the ABA receptors in some plants. However, little is known about the details regarding PYL family genes in the apple (Malusdomestica). In this study, we identified 13 apple PYLs, termed MdPYL1-13, which could be classified into four groups according to structural features of the amino acid sequence. The gene structures and conserved motifs analysis found that the majority of MdPYLs had a similar number of exons and similar conserved motif profile in the same group. In addition, 11 gene pairs were identified to exhibit synteny by synteny analysis between the apple and Arabidopsis. Furthermore, we investigated MdPYLs transcript level in various organs of the red-fleshed apple (Malussieversii f. Neidzwetzkyana (Dieck) Langenf) 'Xinjiang No.1'. The results suggested all MdPYLs within group I were expressed at relatively higher levels in all of the organs tested. However, the genes of group IV had little or no variation. Additionally, we found various hormone and stress-related cis-elements in the promoters of MdPYLs by analyzing cis-elements. Therefore, the expression levels of all MdPYLs were further detected under ABA, PEG, salt, and cold stresses in 'Xinjiang No.1' seedlings. We found that all MdPYLs except for MdPYL11 were upregulated by ABA treatment, 10 genes were upregulated by PEG treatment, 12 genes were upregulated by NaCl treatment, and six genes were upregulated by cold treatment (4 °C) while seven genes were downregulated. Thus, these MdPYLs might be involved in the defense against abiotic stresses. In addition, the interaction between 13 MdPYLs and two 2C protein phosphatases in the apple (MdPP2C65 and MdPP2C72) was investigated in yeast two-hybrid assays. These results suggested that MdPYLs may bind to MdPP2C65 and MdPP2C72 in different manners and with different intensity. Our studies provide useful information for further investigating and researching the regulatory mechanisms of PYL family genes in response to abiotic stresses in the apple.

Translocation of Drought-Responsive Proteins from the Chloroplasts.

Some chloroplast proteins are known to serve as messengers to transmit retrograde signals from chloroplasts to the nuclei in response to environmental stresses. However, whether particular chloroplast proteins respond to drought stress and serve as messengers for retrograde signal transduction are unclear. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) to monitor the proteomic changes in tobacco (Nicotiana benthamiana) treated with drought stress/re-watering. We identified 3936 and 1087 differentially accumulated total leaf and chloroplast proteins, respectively, which were grouped into 16 categories. Among these, one particular category of proteins, that includes carbonic anhydrase 1 (CA1), exhibited a great decline in chloroplasts, but a remarkable increase in leaves under drought stress. The subcellular localizations of CA1 proteins from moss (Physcomitrella patens), Arabidopsis thaliana and rice (Oryza sativa) in P. patens protoplasts consistently showed that CA1 proteins gradually diminished within chloroplasts but increasingly accumulated in the cytosol under osmotic stress treatment, suggesting that they could be translocated from chloroplasts to the cytosol and act as a signal messenger from the chloroplast. Our results thus highlight the potential importance of chloroplast proteins in retrograde signaling pathways and provide a set of candidate proteins for further research.

The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress-induced accumulation of ABA and proline.

Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, is required for many physiological reactions and processes. However, it remains largely unknown how NAD affects plant response to salt stress. We isolated a salt-sensitive mutant named hypersensitive to salt stress (hss) from an ethyl methanesulfonate-induced mutation population. A point mutation was identified by MutMap in the encoding region of Quinolinate Synthase (QS) gene required for the de novo synthesis of NAD. This point mutation caused a substitution of amino acid in the highly-conserved NadA domain of QS, resulting in an impairment of NAD biosynthesis in the mutant. Molecular and chemical complementation have restored the response of the hss mutant to salt stress, indicating that the decreased NAD contents in the mutant were responsible for its hypersensitivity to salt stress. Furthermore, the endogenous levels of abscisic acid (ABA) and proline were also reduced in stress-treated hss mutant. The application of ABA or proline could alleviate stress-induced oxidative damage of the mutant and partially rescue its hypersensitivity to salt stress, but not affect NAD concentration. Taken together, our results demonstrated that the NadA domain of QS is important for NAD biosynthesis, and NAD participates in plant response to salt stress by affecting stress-induced accumulation of ABA and proline.

Heterologous expression of HpBHY and CrBKT increases heat tolerance in Physcomitrella patens.

Heat stress can restrict plant growth, development, and crop yield. As essential plant antioxidants, carotenoids play significant roles in plant stress resistance. ß-carotene hydroxylase (BHY) and ß-carotene ketolase (BKT), which catalyze the conversions of ß-carotene to zeaxanthin and ß-carotene to canthaxanthin, respectively, are key enzymes in the carotenoid biosynthetic pathway, but little is known about their potential functions in stress resistance. Here, we investigated the roles of ß-carotene hydroxylase and ß-carotene ketolase during heat stress in Physcomitrella patens through expressing a ß-carotene ketolase gene from Chlamydomonas reinhardtii (CrBKT) and a ß-carotene hydroxylase gene from Haematococcus pluvialis (HpBHY) in the moss P. patens. In transgenic moss expressing these genes, carotenoids content increased (especially lutein content), and heat stress tolerance increased, with reduced leafy tissue necrosis. To investigate the mechanism of this heat stress resistance, we measured various physiological indicators and found a lower malondialdehyde level, higher peroxidase and superoxide dismutase activities, and higher endogenous abscisic acid and salicylate content in the transgenic plants in response to high-temperature stress. These results demonstrate that CrBKT and HpBHY increase plant heat stress resistance through the antioxidant and damage repair metabolism, which is related to abscisic acid and salicylate signaling.