Expression Profiling of Four Mango FT/TFL1-Encoding Genes under Different Fruit Load Conditions, and Their Involvement in Flowering Regulation.

Plant flowering is antagonistically modulated by similar FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) proteins. In mango (Mangifera indica L.), flowering is induced by cold temperatures, unless the tree is juvenile or the adult tree had a high fruit load (HFL) in the summer. Here, we studied the effects of juvenility and fruit load on the expression of four MiFT/TFL1 genes cloned from the mango 'Shelly' cultivar. Ectopic expression of MiFT1 in Arabidopsis resulted in early flowering, whereas over-expression of MiFT2 and the two cloned MiTFL1 genes repressed flowering. Moreover, juvenility was positively correlated with higher transcript levels of MiFT2 and both MiTFL1s. In trees with a low fruit load, leaf MiFT1 expression increased in winter, whereas HFL delayed its upregulation. MiFT2 expression was upregulated in both leaves and buds under both fruit load conditions. Downregulation of both MITFL1s in buds was associated with a decrease in regional temperatures under both conditions; nevertheless, HFL delayed the decrease in their accumulation. Our results suggest that cold temperature has opposite effects on the expression of MiFT1 and the MiTFL1s, thereby inducing flowering, whereas HFL represses flowering by both suppressing MiFT1 upregulation and delaying MiTFL1s downregulation. The apparent flowering-inhibitory functions of MiFT2 are discussed.

Bumblebees (Bombus terrestris) Improve 'Hass' Avocado (Persea americana) Pollination.

Pollination is limiting for avocado production. We examined whether adding bumblebees (BBs; ca. 10 hives/ha) to conventional honeybees (HB; 5 hives/ha) would improve 'Hass' avocado pollination and yields. A preliminary trial (2017/18) in an avocado orchard with four consecutive rows of 'Hass' followed by one row of 'Ettinger' serving as a pollenizer (20% 'Ettinger') showed a considerable increase in 'Hass' yield in rows adjacent to (up to 80 m from) the BB hives vs. distant rows (=controls). In 2018/19, the trials were extended to three additional orchards. A significant yield increase was obtained in the BB hive-adjacent trees compared to BB hive-distant ones. Similar results were obtained in 2019/20, in experiments conducted throughout the country. The SNP analysis, to determine the parents of 'Hass' fruit at varying distances from the BB hives, showed no differences in the cross-pollination rate ('Hass' × 'Ettinger'). However, pollination rates and the number of germinating pollen grains per stigma decreased with distance from the hives, and correlated to the negative gradient in yield. Taken together, our data suggest that adding BB hives to 'Hass' avocado orchards, at ca. 10 hives/ha resulting in 0.5-1.0 BB visits/tree per min, increases pollination and, accordingly, total yield.

Characterization of Two Ethephon-Induced IDA-Like Genes from Mango, and Elucidation of Their Involvement in Regulating Organ Abscission.

In mango (Mangifera indica L.), fruitlet abscission limits productivity. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide acts as a key component controlling abscission events in Arabidopsis. IDA-like peptides may assume similar roles in fruit trees. In this study, we isolated two mango IDA-like encoding-genes, MiIDA1 and MiIDA2. We used mango fruitlet-bearing explants and fruitlet-bearing trees, in which fruitlets abscission was induced using ethephon. We monitored the expression profiles of the two MiIDA-like genes in control and treated fruitlet abscission zones (AZs). In both systems, qRT-PCR showed that, within 24 h, both MiIDA-like genes were induced by ethephon, and that changes in their expression profiles were associated with upregulation of different ethylene signaling-related and cell-wall modifying genes. Furthermore, ectopic expression of both genes in Arabidopsis promoted floral-organ abscission, and was accompanied by an early increase in the cytosolic pH of floral AZ cells-a phenomenon known to be linked with abscission, and by activation of cell separation in vestigial AZs. Finally, overexpression of both genes in an Atida mutant restored its abscission ability. Our results suggest roles for MiIDA1 and MiIDA2 in affecting mango fruitlet abscission. Based on our results, we propose new possible modes of action for IDA-like proteins in regulating organ abscission.

Genetic diversity of avocado (Persea americana Mill.) germplasm using pooled sequencing.

BACKGROUND: Discovering a genome-wide set of avocado (Persea americana Mill.) single nucleotide polymorphisms and characterizing the diversity of germplasm collection is a powerful tool for breeding. However, discovery is a costly process, due to loss of loci that are proven to be non-informative when genotyping the germplasm. RESULTS: Our study on a collection of 100 accessions comprised the three race types, Guatemalan, Mexican, and West Indian. To increase the chances of discovering polymorphic loci, three pools of genomic DNA, one from each race, were sequenced and the reads were aligned to a reference transcriptome. In total, 507,917 polymorphic loci were identified in the entire collection. Of these, 345,617 were observed in all three pools, 117,692 in two pools, 44,552 in one of the pools, and only 56 (0.0001%) were homozygous in the three pools but for different alleles. The polymorphic loci were validated using 192 randomly selected SNPs by genotyping the accessions within each pool. The sensitivity of polymorphic locus prediction ranged from 0.77 to 0.94. The correlation between the allele frequency estimated from the pooled sequences and actual allele frequency from genotype calling of individual accessions was r = 0.8. A subset of 109 SNPs were then used to evaluate the genetic relationships among avocado accessions and the genetic diversity of the collection. The three races were distinctly clustered by projecting the genetic variation on a PCA plot. As expected, by estimating the kinship coefficient for all the accessions, many of the cultivars from the California breeding program were closely related to each other, especially, the Hass-like ones. The green-skin avocados, e.g., 'Bacon', 'Zutano', 'Ettinger' and 'Fuerte' were also closely related to each other. CONCLUSIONS: A framework for SNP discovery and genetically characterizing of a breeder's accessions was described. Sequencing pools of gDNA is a cost-effective approach to create a genome-wide stock of polymorphic loci for a breeding program. Reassessing the botanical and the genetic knowledge about the germplasm accessions is valuable for future breeding. Kinship analysis may be used as a first step in finding a parental candidates in a parentage analyses.

Distinct organ-specific and temporal expression profiles of auxin-related genes during mango fruitlet drop.

In mango, fruitlet abscission initiates with a decrease in polar auxin transport through the abscission zone (AZ), triggered by ethylene. To explore the molecular components affecting this process, we initially conducted experiments with developing fruitlet explants in which fruitlet drop was induced by ethephon, and monitored the expression patterns of distinct indole-3-acetic acid (IAA)-related genes, comparing control vs. ethephon-treated pericarp and AZ profiles. Over the examined time period (48 h), the accumulation of MiPIN1 and MiLAX2 IAA-efflux and influx genes decreased in both control and treated tissues. Nevertheless, ethephon-treated tissues displayed significantly lower levels of these transcripts within 18-24 h. An opposite pattern was observed for MiLAX3, which overall exhibited up-regulation in treated fruitlet tissues. Ethephon treatment also induced an early and pronounced down-regulation of five out of six IAA-responsive genes, and a substantial reduction in the accumulation of two IAA-synthesis related transcripts, contrasting with significant up-regulation of Gretchen Hagen3 transcript (MiGH3.1) encoding an IAA-amino synthetase. Furthermore, for both control and treated AZ, the decrease in IAA-carrier transcripts was associated with a decrease in IAA content and an increase in IAA-Asp:IAA ratio, suggesting that fruitlet drop is accompanied by formation of this non-hydrolyzed IAA-amino acid conjugate. Despite these similarities, ethephon-treated AZ displayed a sharper decrease in IAA content and higher IAA-Asp:IAA ratio within 18 h. Lastly, the response of IAA-related genes to exogenous IAA treatment was also examined. Our results are discussed, highlighting the roles that distinct IAA-related genes might assume during mango fruitlet drop.

Expression profiling of FLOWERING LOCUS T-like gene in alternate bearing 'Hass' avocado trees suggests a role for PaFT in avocado flower induction.

In many perennials, heavy fruit load on a shoot decreases the ability of the plant to undergo floral induction in the following spring, resulting in a pattern of crop production known as alternate bearing. Here, we studied the effects of fruit load on floral determination in 'Hass' avocado (Persea americana). De-fruiting experiments initially confirmed the negative effects of fruit load on return to flowering. Next, we isolated a FLOWERING LOCUS T-like gene, PaFT, hypothesized to act as a phloem-mobile florigen signal and examined its expression profile in shoot tissues of on (fully loaded) and off (fruit-lacking) trees. Expression analyses revealed a strong peak in PaFT transcript levels in leaves of off trees from the end of October through November, followed by a return to starting levels. Moreover and concomitant with inflorescence development, only off buds displayed up-regulation of the floral identity transcripts PaAP1 and PaLFY, with significant variation being detected from October and November, respectively. Furthermore, a parallel microscopic study of off apical buds revealed the presence of secondary inflorescence axis structures that only appeared towards the end of November. Finally, ectopic expression of PaFT in Arabidopsis resulted in early flowering transition. Together, our data suggests a link between increased PaFT expression observed during late autumn and avocado flower induction. Furthermore, our results also imply that, as in the case of other crop trees, fruit-load might affect flowering by repressing the expression of PaFT in the leaves. Possible mechanism(s) by which fruit crop might repress PaFT expression, are discussed.

PaKRP, a cyclin-dependent kinase inhibitor from avocado, may facilitate exit from the cell cycle during fruit growth.

Previous studies using 'Hass' avocado cultivar showed that its small-fruit (SF) phenotype is limited by cell number. To explore the molecular components affecting avocado cell production, we isolated four cDNAs encoding: an ICK/KRP protein, known to play cell cycle-regulating roles through modulation of CDK function; two CDK proteins and a D-type cyclin, and monitored their expression patterns, comparing NF (normal fruit) versus SF profiles. The accumulation of PaKRP gradually deceased during growth in both fruit populations. Despite these similarities, SF exhibited higher levels of PaKRP accumulation at early stages of growth. Moreover, in NF, augmented PaKRP expression coincided with a decrease in CDK and PaCYCD1 levels, whereas in SF, enhanced PaKPR expression was coupled with an earlier decline of CDK and PaCYCD1 levels. For both NF and SF, enhanced mesocarp PaKRP transcript accumulation, was associated with elevated abscisic acid (ABA) and ABA catabolites content. Nevertheless, the collective ABA levels, including catabolites, were substantially higher in SF tissues, as compared with NF tissues. Finally, additional expression analysis revealed that in cultured cells, PaKRP could be induced by ABA. Together, our data links PaKRP with exit from the fruit cell cycle and suggest a role for ABA in controlling its expression.