CRISPR genome editing has shown tremendous potential in genetic improvement of citrus. So far, citrus genome editing has been conducted using juvenile tissues resulting in genome-edited citrus plants that require multiple years before they can produce flowers and fruit. Here we tested whether citrus genome editing via mature tissue transformation can overcome such a hurdle. CsLOB1 is a susceptibility gene for citrus canker caused by Xanthomonas citri subsp. citri (Xcc). The transcription activator-like effector PthA4 of Xcc activates CsLOB1 by binding to the effector-binding element in its promoter (EBEpthA4-CsLOBP). In Valencia sweet orange, two CsLOB1 promoter alleles are present: TI CsLOBP, and TII CsLOBP. We specifically utilized a CRISPR/Cas9 construct (GFP-p1380N-Cas9/sgRNA: CsLOBP2) targeting EBEpthA4 in TI CsLOBP but not TII CsLOBP to test genome editing efficacy and sgRNA: CsLOBP2-targeting specificity. GFP-p1380N-Cas9/sgRNA: CsLOBP2 function was first validated using Xcc-facilitated agroinfiltration in Valencia leaves. The construct was subsequently introduced into Valencia mature internodal stem segments via Agrobacterium-mediated transformation, generating three independent transgenic lines (#V2, #V3 and #V5). Targeted mutations in EBEpthA4-TI CsLOBP were detected in all three lines with mutation frequencies of 100%, 21.43% and 41.94% in #V2, #V3 and #V5, respectively, while no mutations were detected in TII CsLOBP. Infection with XccΔpthA4:dCsLOB1.3, carrying a designer TALE that specifically activates TI CsLOBP, resulted in reduced canker symptoms in #V2. Importantly, all three EBEpthA4-TI CsLOBP edited lines flowered within 15 months. Finally, no off-target mutations were identified for Cas9/sgRNA: CsLOBP2. In sum, these results demonstrate that CRISPR/Cas9-mediated genome modification through mature citrus transformation can achieve high genome editing efficacy and overcome the juvenility.

Citrus greening disease, or Huanglongbing (HLB), has caused devastating losses to citrus production in Florida, with yields declining by over 90% since 2005. Despite extensive efforts, no sustainable solution has been widely effective. Here, transgenic 'Hamlin' sweet orange lines engineered to constitutively express the Arabidopsis NPR1 (AtNPR1) gene, a key regulator of systemic acquired resistance, are evaluated for health and environmental risks. These citrus lines exhibit strong HLB tolerance, with reduced disease symptoms, sustained fruit production, and no apparent negative phenotypic abnormalities. Comprehensive risk assessment reveals minimal exposure, health, or environmental risk. The AtNPR1 protein is: (1) barely detectable in fruit, (2) rapidly degraded in simulated gastrointestinal fluids, and (3) not similar to known allergens or toxins. Whole-genome sequencing identified the T-DNA insertion sites as heterozygous in either chromosome 1 or 6, with no disruptions in known fruit-producing genes. PCR markers were developed for rapid line identification. The selected lines are currently in a small field trial under high HLB pressure and continue to exhibit low visual HLB symptoms and positive horticultural traits. These findings support the initial requirements for regulatory approval of these transgenic citrus varieties, offering a promising strategy for sustainable citrus production.

Pepper anthracnose is a globally devastating fungal disease caused by Colletotrichum spp. In this study, we explored the molecular mechanisms underlying anthracnose resistance in Capsicum annuum by comparing a resistant variety 225 with a susceptible variety 307. Phenotypic analysis revealed that variety 225 displayed stronger resistance than variety 307. Through comparative transcriptome analysis and weighted gene co-expression network analysis (WGCNA), 17 gene modules were identified, among which the salmon module showed a strong association with resistance in variety 225. Within this module, 18 hub genes—including Ca59V2g00372.1 (CaTLP6), encoding a thaumatin-like protein (TLP)—were significantly upregulated upon infection. A genome-wide analysis identified 31 CaTLP genes in C. annuum, with members of group V (such as CaTLP6) exhibiting induced expression post-inoculation of Colletotrichum scovillei. Subcellular localization analysis indicated that group V CaTLP proteins were associated with the plasma membrane, suggesting a role in pathogen recognition. These findings highlight the significance of CaTLP genes, particularly those in group V, in pepper’s defense against anthracnose caused by C. scovillei and offer promising targets for breeding resistant cultivars.

Summary Plants adjust the size of their stomatal openings to balance CO 2 intake and water loss. Carbonic anhydrases (CAs) facilitate the conversion between CO 2 and HCO 3 − , and the OsβCA1 mutant in rice ( Oryza sativa ) shows similar traits in carbon fixation and stomatal response to CO 2 as the dual βCA mutants in Arabidopsis thaliana . However, the exact role of OsβCA1 in these processes was unclear. We used gene editing, molecular biology, and plant physiology to study how OsβCA1 contributes to carbon fixation, stomatal opening, and CO 2 responses. OsβCA1 produces three isoforms (OsβCA1A, OsβCA1B, and OsβCA1C) through alternative transcriptional initiation, which localize to the chloroplast, cell membrane, and cytosol, respectively. Protein measurements revealed that OsβCA1A/C and OsβCA1B contribute 97 and 3% to OsβCA1, respectively. By creating specific mutants for each isoform, our results found that the chloroplast and cell membrane isoforms independently participate in carbon fixation and regulation of stomatal aperture. Furthermore, the complete knockout of OsβCA1 caused a delayed response to low CO 2 . Our findings provide new insights into the generation and function of different OsβCA1 isoforms, clarifying their roles in CO 2 diffusion, CO 2 fixation and stomatal regulation in rice.

Background: Citri Reticulatae Pericarpium (CRP), a classic Chinese medicinal herb rich in bioactive compounds, exhibits antioxidant, anti-inflammatory, anticancer, and cardiovascular protective activities. The ethnopharmacological properties of CRP methanol extract (CRPME) may contribute to its efficacy in asthma management. This study investigates the therapeutic effects of CRPME on ovalbumin (OVA)-induced asthma in mice and elucidates its underlying mechanisms using in vivo experiments combined with network pharmacology analysis. Materials: The major chemical constituents of CRPME were identified via high-performance liquid chromatography (HPLC). We evaluated the impact of CRPME on airway inflammation in OVA-induced asthmatic mice and employed network pharmacology to predict its antiasthmatic mechanisms, providing insights into the molecular basis of CRP's therapeutic potential in asthma. Results: CRPME significantly alleviated OVA-induced asthmatic symptoms and lung pathological damage, while reducing interleukin (IL)-4, IL-13, and IL-17 levels in bronchoalveolar lavage fluid (BALF) and serum-specific immunoglobulin E (IgE) levels. Network pharmacology analysis revealed that CRPME may exert antiasthmatic effects by regulating IL-17, hypoxia-inducible factor 1 (HIF-1), nuclear factor-κB (NF-κB), and advanced glycation end-product receptor (AGE-RAGE) signaling pathways through active constituents including naringin, hesperidin, demethoxy hesperidin, luteolin, and nobiletin. Conclusion: Integrating experimental and network pharmacology data, this study demonstrates that bioactive compounds in CRPME mitigate allergic responses in asthmatic mice, supporting its potential as a therapeutic agent for allergic asthma.

Advances in carbohydrate metabolism prompted its essential role in defense priming and sweet immunity during plant-pathogen interactions. Nevertheless, upstream responding enzymes in the sucrose metabolic pathway and associated carbohydrate derivatives underlying fungal pathogen challenges remain to be deciphered in Populus, a model tree species. In silico deduction of genomic features, including phylogenies, exon/intron distributions, cis-regulatory elements, and chromosomal localization, identified 59 enzyme genes (11 families) in the Populus genome. Spatiotemporal expression of the transcriptome and the quantitative real-time PCR revealed a minuscule number of isogenes that were predominantly expressed in roots. Upon the pathogenic Fusarium solani (Fs) exposure, dynamic changes in the transcriptomics atlas and experimental evaluation verified Susy (PtSusy2 and 3), CWI (PtCWI3), VI (PtVI2), HK (PtHK6), FK (PtFK6), and UGPase (PtUGP2) families, displaying promotions in their expressions at 48 and 72 h of post-inoculation (hpi). Using the gas chromatography-mass spectrometry (GC–MS)-based non-targeted metabolomics combined with a high-performance ion chromatography system (HPICS), approximately 307 metabolites (13 categories) were annotated that led to the quantification of 46 carbohydrates, showing marked changes between three compared groups. By contrast, some sugars (e.g., sorbitol, L-arabitol, trehalose, and galacturonic acid) exhibited a higher accumulation at 72 hpi than 0 hpi, while levels of α-lactose and glucose decreased, facilitating them as potential signaling molecules. The systematic overview of multi-omics approaches to dissect the effects of Fs infection provides theoretical cues for understanding defense immunity depending on fine-tuned Suc metabolic gene clusters and synergistically linked carbohydrate pools in trees.

The Plateau Hongliu (Myricaria elegans Royle.) is a woody shrub halophyte that thrives in arid areas of western Tibet, in the Himalayan Mountains. It is acclaimed as superior in saline stress acclimation and as a critical pharmaceutical resource of the Tibetan traditional herb. Nevertheless, the mitogenome in the genus Myricaria remains unknown. Here, using the Illumina and PacBio sequencing assays, the first complete mitogenome of the M. elegans revealed a multi-branched skeleton with a total length of 416,354 bp and GC content of 44.33%, comprising two circular molecules (M1 and 2). The complete mitogenome annotates 31 unique protein-encoding genes (PEGs), fifteen tRNAs, and three rRNA genes. The UAA exhibits the most prominent codon usage preference as a termination, followed by UUA codons for leucine. The mitogenome contains 99 simple sequence repeats and 353 pairs of dispersed repeats, displaying the most frequent in palindromic repeats. Gene transfer analyses identified 8438 bp of 18 homologous fragments from the plastome, accounting for 2.03% of the total length. Using the PREP suite, 350 C-U RNA editing sites were predicted, of which nad4 and ccmB were on the top frequency. Syntenic and phylogenetic analyses suggested weakly conserved patterns of M. elegans in Caryophyllales owing to the genome rearrangement. In summary, the deciphered unique features and complexities of the mitogenome in M. elegans provide novel insights into understanding the evolution and biological conservation underlying climate resilience in halophytes.

Transformation efficiencies of sweet orange cultivars 'Florida EV1' and 'Valencia', recalcitrant to Agrobacterium transformation, were investigated using liquid culture in We-VTM vessels. The two mature cultivars were transformed using Agrobacterium with a vector containing selectable markers, npt II and TIPS-EPSPS, and the GFP reporter. Transgenics were identified with GFP in liquid culture at 0, 100, and 200 mgL-1 kanamycin or in the semi-solid control with 100 mgL-1 kanamycin. For 'Florida EV1', there were significant differences in the mean transformation efficiency based on the number of shoots screened (TES) at all kanamycin concentrations. Selection at 200 mgL-1 was better than at lower concentrations in liquid or semi-solid control medium with 100 mgL-1 kanamycin. The variable TEE, transformation efficiency based on the number of explants, did not discern differences. The means +/- standard errors for TES at 200 mgL-1 were 7.9% +/- 2.7% for 'Florida EV1' and 2.4% +/- 1.7% for 'Valencia'. In total, 74 transgenics were produced in 'Florida EV1', whereas seven were generated in 'Valencia'. Obtaining transgenics in 'Florida EV1' was easy; fewer shoots were screened at 200 mgL-1.'Florida EV1' exhibited better regeneration ability, and all transgenics survived on glyphosate medium, suggesting the TIPS-EPSPS selectable marker could be useful in transformation. Molecular analyses confirmed their transgenic nature. 'Florida EV1' trees produced fruit earlier than 'Valencia' in less than two years. 'Florida EV1' could accelerate the production of HLB disease-resistant trees.

Arrow bamboo (Fargesia qinlingensis) is endemic to the Qinling Mountains and has remarkable adaptive resilience to changing climates. However, its complete mitogenome remains unknown. Using the Illumina and PacBio HiFi sequencing platforms, we found that the mitogenome assembly of the F. qinlingensis has a multi-branched skeleton comprising three linear molecules (M1, M2, and M3), with a length of 442,368 bp and a GC content of 44.05%. Thirty-five unique PCGs were identified in the complete mitogenome, including twenty-four core structural genes, eleven noncore structural genes, three rRNAs, and sixteen tRNAs. The GCU for alanine and CAA for glutamine represented the most significant frequency (RSCU = 1.55) in the codon usage preference. A total of 51, 28, and 14 SSRs were determined on M1, M2, and M3, respectively. The mitogenome contained 149 pairs of dispersed repeats with lengths greater than 30 bp, the most abundant of which were 82 forward and 67 palindromic repeats. A long repeat sequence (14,342 bp) was characterized in mediating mitogenome recombination. DNA transfer analyses suggested that 44 MTPTs (30,943 bp, 6.99%) originated from the plastome. Among the 482 potential C-U/T RNA-editing sites predicted in 35 PCGs, ccmFn (38 times) and ccmC (36 times) shoed the highest frequency. Collinearity and phylogenetic trees revealed the close relationship between F. qinlingensis and Bambusa oldhamii. The primary features of the mitogenome of F. qinlingensis will help decipher the functional mitochondrial traits related to growth performance and climate resilience. Moreover, our findings provide insights into the evolution, environmental adaptation, and sustainable use of subalpine bamboo resources in the Qinling Mountains.

Integration of the pectin homogalacturonan (HG) integrity and remodeling into defense immunity facilitates building blocks of stress resilience within the host and pathogen interactions in the forests. Recent advances in apoplastic pectin methylesterases (PMEs) and coordinated PME inhibitors (PMEIs) deployed immune signaling, highlighting their critical roles in regulating biotic stress adaptation. Nevertheless, the PME and PMEI multiple isoenzyme families involved in defense priming in the model tree species are largely unknown. The in silico demonstration of the molecular profile identified 70 PME- (50 type-I proPME and 20 type-II PME) and 49 PMEI-encoding genes in the Populus genome. Evaluation of the transcript abundance verified 20 PtproPMEs and 18 PtPMEIs that showed predominant expression in the root and genetic redundancy for type-II PtPMEs. Dynamic changes in the transcriptome and experimental evaluation revealed a minuscule number of type-I proPMEs and PMEIs that showed marked promotions during a time-course Fusarium solani (Fs) infection. Using GC/LC-mass-based non-targeted metabolomics to inspect structure-related carbohydrates and PME activities suggested a correlation between proPME-PMEI co-expression and deformed pectin HG biosynthesis. Further functional examination of the enzyme inhibitory affinities of recombinant PtPMEI1 and 39 in vitro and apoplastic patterns suggested a clustering of the type-I proPME in the regulation of the innate immunity by maintaining homeostasis of the pectin HG de-methylesterification (DME) in the cell wall. The coherent omics-wide survey of the fungi pathogen-induced PME-PMEIs and disturbed carbohydrate accumulation provide theoretical cues for in-depth mining of the biological significance of the underlying immune signaling networks within an apoplastic niche in trees.

The CDPK family genes play crucial roles in signal transduction pathways during plant development and stress response. In this study, we comprehensively analyzed the CDPK family genes in mustard (Brassica juncea L.), resulting in the identification of 101 genes (BjuCDPK1–101) located on chromosomes AA_Chr01 to BB_Chr08. RNA-seq analysis showed that most BjuCDPK genes were predominantly expressed in the root and flower bud, suggesting their organ-specific expression patterns. Furthermore, the expression levels of most BjuCDPK genes were significantly altered by cold stress. The promoter regions of BjuCDPK genes had abundant cis-acting elements related to hormones and stress. The interaction network analysis predicted that BjuCDPK proteins respond to abiotic stress probably via interaction with ABF1, ABF4, and/or DI19. Our findings provide valuable information for further the investigation of the cold stress adaption of mustard via the CDPK signaling pathway.

Annona is a genus of family Annonaceae within the magnoliids and plays a crucial role in revealing the evolution of magnolias. Annona species provide important fruit resources. Here, we report a chromosome-level genome assembly of A. montana, an edible and ornamental fruit species. Integration with other genomes provides clear evidence that the magnoliids were sisters to eudicots, and the ASTRAL trees showed discordance in the phylogenetic position of magnoliids, which might be caused by incomplete lineage sorting (ILS). Whole genome duplication (WGD) analysis showed that the common ancestor of A. montana and Liriodendron chinense experienced a WGD event, and this WGD event occurred after the splitting of Magnoliales and Laurales. We identified the gene family expansions and contractions in Annonaceae. Based on the identification of MADS-box gene families, we inferred the pathway integrators of morphological regulation, the occurrence of florescence and the development of fruit in A. montana. In addition, we identified key sugar transporter genes and the key enzyme genes related to sugar accumulation in A. montana fruit. The gene function analysis indicated that starch and cell wall degradation might be the main reasons for the softening of A. montana fruit. Furthermore, aromatic alcohols were suggested be the main volatile aromatic compounds in A. montana fruit. Our results provide the genetic basis of fruit development, softening, aroma, and sugar accumulation in A. montana and the evolution and diversification of Annonaceae.

Gravity Wells are attachments for We-V liquid culture vessels designed to gradually dispense liquid medium into the vessels during in vitro plant propagation. The objective of this study was to compare Agrobacterium-mediated mature citrus rootstock selection in We-V vessels with Gravity Wells to the standard transformation protocol using semi-solid medium and to determine which kanamycin concentrations (0, 50, 100, 150, 200 mg/l) were best for liquid selection. Two mature citrus rootstocks were each transformed with a binary vector: Kuharske rootstock was transformed with pGNA, and US-942 rootstock was transformed with pGNS. The in vitro plant growth period was for a total of seven weeks, in which the explants were incubated in the dark on semi-solid medium for three weeks, followed by four weeks in the light in liquid or semi-solid medium. In total, 108 Kuharske transgenics and 40 US-942 transgenics were obtained over all treatments. The two cultivars were analyzed separately using ANOVAs. The response variables analyzed were the mean number of shoots longer than 2 mm (MSL>2), the mean transformation efficiency based on the number of explants (TEE), and the mean transformation efficiency based on the number of screened shoots (TES). For Kuharske and US-942, there were significant differences among kanamycin concentrations and between liquid vs semi-solid medium with liquid medium being superior to semi-solid medium because of an increased TES while also lowering MSL>2. The TEE variable was also significant for Kuharske, but it was not for US-942. US-942 formed fewer organogenic shoots in all other treatments. Selection at higher kanamycin concentrations (150 and 200 mg/l) yielded more transgenics and fewer shoots had to be screened using We-V vessels with gravity wells. PCR confirmed the presence of the transgenes, and Southern blots showed integration of the nptII transgene into the genome.

Two Citrus sinensis (L.) phosphomannose isomerase (PMI) genes, CsPMI1 and CsPMI2, were evaluated as novel selectable markers in mature citrus transformation. Transgenic shoots produced after transformation of Kuharske rootstock with each PMI construct were selected on six treatments of mannose and sucrose. For CsPMI1, there were no significant differences among the various mannose and sucrose treatments for the mean number of positive shoots (PS), the mean transformation efficiency based on the number of shoots (TES), or the mean transformation efficiency based on the number of explants (TEE). However, for the CsPMI2 gene, the number of transgenics produced in two treatments (7.5 g L−1 mannose + 22.5 g L−1 sucrose and 15 g L−1 mannose + 15 g L−1 sucrose) was significantly greater than the sucrose control for TES at 4.2% and 3.7%, respectively. Moreover, TEE at 4.2% in the 15 g L−1 mannose + 15 g L−1 sucrose treatment, supported the TES value. Most of the transgenic lines demonstrated higher in vivo and in vitro enzyme assays compared with the wild-type control. CsPMI2 provided acceptable selection in mature citrus, and it will be applied in future intragenic research.

Integrating amino acid metabolic pathways into plant defense and immune systems provides the building block for stress acclimation and host-pathogen interactions. Recent progress in L-aspartate (Asp) and its deployed metabolic pathways highlighted profound roles in plant growth and defense modulation. Nevertheless, much remains unknown concerning the multiple isoenzyme families involved in Asp metabolic pathways in Populus trichocarpa, a model tree species. Here, we present comprehensive features of 11 critical isoenzyme families, representing biological significance in plant development and stress adaptation. The in silico prediction of the molecular and genetic patterns, including phylogenies, genomic structures, and chromosomal distribution, identify 44 putative isoenzymes in the Populus genome. Inspection of the tissue-specific expression demonstrated that approximately 26 isogenes were expressed, predominantly in roots. Based on the transcriptomic atlas in time-course experiments, the dynamic changes of the genes transcript were explored in Populus roots challenged with soil-borne pathogenic Fusarium solani (Fs). Quantitative expression evaluation prompted 12 isoenzyme genes (PtGS2/6, PtGOGAT2/3, PtAspAT2/5/10, PtAS2, PtAspg2, PtAlaAT1, PtAK1, and PtAlaAT4) to show significant induction responding to the Fs infection. Using high-performance liquid chromatography (HPLC) and non-target metabolomics assay, the concurrent perturbation on levels of Asp-related metabolites led to findings of free amino acids and derivatives (e.g., Glutamate, Asp, Asparagine, Alanine, Proline, and α-/γ-aminobutyric acid), showing marked differences. The multi-omics integration of the responsive isoenzymes and differential amino acids examined facilitates Asp as a cross-talk mediator involved in metabolite biosynthesis and defense regulation. Our research provides theoretical clues for the in-depth unveiling of the defense mechanisms underlying the synergistic effect of fine-tuned Asp pathway enzymes and the linked metabolite flux in Populus.

Valine-glutamine (VQ) motif-containing proteins are a class of highly conserved transcriptional regulators in plants and play key roles in plant growth, development, and response to various stresses. However, the VQ family genes in mustard have not yet been comprehensively identified and analyzed. In this study, a total of 120 VQ family genes (BjuVQ1 to BjuVQ120), which were unevenly distributed on 18 chromosomes (AA_Chr01 to BB_Chr08), were characterized in mustard. A phylogenetic tree analysis revealed that the BjuVQ proteins were clustered into nine distinct groups (groups I to IX), and members in the same group shared a highly conserved motif composition. A gene structure analysis suggested that most BjuVQ genes were intronless. A gene duplication analysis revealed that 254 pairs of BjuVQ genes were segmentally duplicated and one pair was tandemly duplicated. Expression profiles obtained from RNA-seq data demonstrated that most BjuVQ genes have different gene expression profiles in different organs, including leaf, stem, root, flower bud, pod, and seed. In addition, over half of the BjuVQ genes were differentially expressed at some time points under low temperature treatment. The qRT-PCR data revealed that BjuVQ23, BjuVQ55, BjuVQ57, BjuVQ67, BjuVQ100, and BjuVQ117 were upregulated in response to cold stress. Taken together, our study provides new insights into the roles of different BjuVQ genes in mustard and their possible roles in growth and development, as well as in response to cold stress.

Key message Three new artificial microRNA vectors were constructed and evaluated, and results showed that these vectors are highly efficient in the silencing of the citrusPHYTOENE DESATURASEgene.

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 ( AtSUC2 ) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30–40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1 ) and APETALA1 ( AP1 ) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR ( AP2 / ERF ) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

SWEET/MtN3/saliva genes are prevalent in cellular organisms and play diverse roles in plants. These genes are widely considered as evolutionarily conserved genes, which is inconsistent with their extensive expansion and functional diversity. In this study, SWEET genes were identified from 31 representative plant species, and exhibited remarkable expansion and diversification ranging from aquatic to land plants. Duplication detection indicated that the sharp increase in the number of SWEET genes in higher plants was largely due to tandem and segmental duplication, under purifying selection. In addition, phylogeny reconstruction of SWEET genes was performed using the maximum-likelihood (ML) method; the genes were grouped into four clades, and further classified into 10 monocot and 11 dicot subfamilies. Furthermore, selection pressure of SWEET genes in different subfamilies was investigated via different strategies (classical and Bayesian maximum likelihood (Datamonkey/PAML)). The average dN/dS for each group were lower than one, indicating purifying selection. Individual positive selection sites were detected within 4 of the 21 sub-families by both two methods, including two monocot subfamilies in Clade III, harboring five rice SWEET homologs characterized to confer resistance to rice bacterial blight disease. Finally, we traced evolutionary fate of SWEET genes in clade III for functional characterization in future.