Altered sensitivity to ethylene in ‘Tardivo’, a late‐ripening mutant of Clementine mandarin

‘Tardivo’ mandarin is a mutant of ‘Comune’ Clementine with a delay in peel degreening and coloration, allowing late harvesting. In this work, we have explored if the late‐harvesting phenotype of ‘Tardivo’ mandarin is related to altered perception and sensitivity to ethylene. The peel degreening rate was examined after a single ethephon treatment or during a continuous ethylene application in fruits at two maturation stages. In general, ethylene‐induced peel degreening was considerably delayed and reduced in fruits of ‘Tardivo’, as well as the concomitant reduction of chlorophyll (Chl) and chloroplastic carotenoids, and the accumulation of chromoplastic carotenoids. Analysis of the expression of genes involved in Chl degradation, carotenoids, ABA, phenylpropanoids and ethylene biosynthesis revealed an impairment in the stimulation of most genes by ethylene in the peel of ‘Tardivo’ fruits with respect to ‘Comune’, especially after 5 days of ethylene application. Moreover, ethylene‐induced expression of two ethylene receptor genes, ETR1 and ETR2, was also reduced in mutant fruits. Expression levels of two ethylene‐responsive factors, ERF1 and ERF2, which were repressed by ethylene, were also impaired to a different extent, in fruits of both genotypes. Collectively, results suggested an altered sensitivity of the peel of ‘Tardivo’ to ethylene‐induced physiological and molecular responses, including fruit degreening and coloration processes, which may be time‐dependent since an early moderated reduction in the responses was followed by the latter inability to sustain ethylene action. These results support the involvement of ethylene in the regulation of at least some aspects of peel maturation in the non‐climacteric citrus fruit.     

Probing the role of endogenous ethylene in the degreening of citrus fruit with ethylene antagonists

The ethylene antagonists, 2,5-norbornadiene (NBD) and silver nitrate, were used to probe the involvement of endogenous ethylene in the natural degreening of citrus fruit. Mature-green, detached Shamouti orange (Citrus sinensis L. Osbeck) fruit were treated with NBD vapor or dipped in solutions of silver nitrate. More than 80% of the chlorophyll was lost from control fruit after 8 days. NBD (0.11 mmole/liter) inhibited the loss of chlorophyll by 60%. NBD also antagonized the degreening induced by exogenous ethylene by 50%. Silver nitrate (0.1 mM) inhibited the loss of chlorophyll by 55%. Ethylene evolution of mature, green detached fruit was <2 nl.fruit^-1.h^-1 (ca. 13.5 nl.Kg^-1FW.h^-1) and did not change significantly for 7 days after harvest. NBD concentrations up to 0.22 mmole/liter did not enhance ethylene evolution. Not with-standing the extremely low amounts of ethylene evolved, the inhibition of degreening by NBD and silver nitrate suggests that endogenous ethylene is involved in the control of this process in mature citrus fruit.     

Involvement of ethylene in chlorophyll degradation in peel of citrus fruits

The effect of ethylene on chlorophyll degradation in the peel of Robinson tangerine (X Citrus reticulata Blanco) and calamondin (X Citrofortunellamitis [Blanco] Ingram and Moore) fruits was studied. The chlorophyll degrading system in the peel of these two citrus species was not self-sustaining but required ethylene to function. Chlorophyll degradation ceased immediately when fruit were removed from ethylene and held in ethylene-free air at 0.2 atmospheric pressure. However, at atmospheric pressure, chlorophyll degradation continued for 24 hours in the absence of exogenous ethylene. Although chlorophyllase levels were negatively correlated with chlorophyll content in the peel (r = −0.981; P < 0.01), the level of chlorophyllase activity did not change when fruit were removed from ethylene, even though chlorophyll degradation had stopped. From these observations, it was concluded that ethylene is necessary for chlorophyll degradation in the two species of citrus studied, but its primary role is not solely for the induction of chlorophyllase activity.     

Sugar regulation of plastid interconversions in epicarp of citrus fruit

Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.

The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO₃ in the media.

It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening.

     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

microRNA159‐targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA‐based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and ‐2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159‐overexpressing tomato cv. Micro‐Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159‐overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA‐like genes and the miR167/SlARF8a module. Similarly to SlMIR159‐overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159‐targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.     

Conversion of chlorophyll b to chlorophyll a precedes magnesium dechelation for protection against necrosis in Arabidopsis

Chlorophyll is a deleterious molecule that generates reactive oxygen species and must be converted to non‐toxic molecules during plant senescence. The degradation pathway of chlorophyll a has been determined; however, that of chlorophyll b is poorly understood, and multiple pathways of chlorophyll b degradation have been proposed. In this study, we found that chlorophyll b is degraded by a single pathway, and elucidated the importance of this pathway in avoiding cell death. In order to determine the chlorophyll degradation pathway, we first examined the substrate specificity of 7‐hydroxymethyl chlorophyll a reductase. 7‐hydroxymethyl chlorophyll a reductase reduces 7‐hydroxymethyl chlorophyll a but not 7‐hydroxymethyl pheophytin a or 7‐hydroxymethyl pheophorbide a. These results indicate that the first step of chlorophyll b degradation is its conversion to 7‐hydroxymethyl chlorophyll a by chlorophyll b reductase, although chlorophyll b reductase has broad substrate specificity. In vitro experiments showed that chlorophyll b reductase converted all of the chlorophyll b in the light‐harvesting chlorophyll a/b protein complex to 7‐hydroxymethyl chlorophyll a, but did not completely convert chlorophyll b in the core antenna complexes. When plants whose core antennae contained chlorophyll b were incubated in the dark, chlorophyll b was not properly degraded, and the accumulation of 7‐hydroxymethyl pheophorbide a and pheophorbide b resulted in cell death. This result indicates that chlorophyll b is not properly degraded when it exists in core antenna complexes. Based on these results, we discuss the importance of the proper degradation of chlorophyll b.     

Expression of a senescence-associated cysteine protease gene related to peel pitting of navel orange (Citrus sinensis L. Osbeck)

Previously, a suppression subtractive hybridization library was constructed to identify differentially expressed genes in peel pitting of navel orange fruit and a cDNA fragment sharing high similarities to cysteine protease genes was identified. In this study, we cloned its full-length cDNA sequence, designated CsCP, using the Rapid amplification of cDNA ends approach. It consists of 1,409 nucleotides and its ORF encodes 361 amino acids predicted to have an N-terminal signal peptide. Phylogenetic analysis revealed that CsCP belonged to the aleurain group in papain family of cysteine proteases. According to quantitative RT-PCR, the expression of CsCP was enhanced during the development of postharvest peel pitting concomitant with senescence, although it was detectable in all tested tissues including root, leaf, flower and peel of fruit. RNA gel blot analysis showed that the CsCP expression was induced by hypoxia (3% O₂), but repressed by anoxia (0% O₂), wounding, ethylene and high temperature (40°C). Conclusively, the CsCP is a senescence-associated gene and up-regulated during the development of citrus postharvest peel pitting, which provides a basis to understand its role in citrus peel pitting.     

Genome-wide identification and expression analysis of MATE gene family in citrus fruit (Citrus clementina)

Multidrug and toxic compound extrusion (MATE) proteins are a class of secondary active multidrug transporters. In plants, this family has significantly expanded and is involved in numerous plant physiological processes. Although MATE proteins have been identified in an increasing number of species, the understanding about this family in citrus remains unclear. In this study, a total of 69 MATE transporters were identified in the citrus genome (Citrus clementina) and classified into four groups by phylogenetic analysis. Tandem and segmental duplication events were the main causes of the citrus MATE family expansion. RNA-seq and qRT-PCR analyses were performed during citrus fruit development. The results indicated that CitMATE genes showed specific expression profiles in citrus peels and flesh at different developmental stages. Combined with the variations of flavonoids and citrate levels in citrus fruit, we suggested that CitMATE43 and CitMATE66 may be involved in the transport process of flavonoids and citrate in citrus fruit, respectively. In addition, two flavonoids positive regulators, CitERF32 and CitERF33, both directly bind to and activated the CitMATE43 promoter. Our results provide comprehensive information on citrus MATE genes and valuable understanding for the flavonoids and citrate metabolism in citrus fruit.     

Field evaluation of the relative attractiveness of enriched ginger root oil (EGO)lure and trimedlure for African Ceratitis species (Diptera: Tephritidae)

The males of some fruit flies (Diptera: Tephritidae) are known to be attracted to specific parapheromones. The trapping results between trimedlure (TML) and enriched ginger root oil (EGOlure) were compared at two experimental sites in Morogoro (Central Tanzania) for a period of 12 weeks co‐inciding with the main citrus season. Both attractants captured a comparable diversity of fruit flies, except that EGOlure also attracted fruit flies, such as Ceratitis cosyra, not normally found in TML‐baited traps. Both EGOlure and TML attracted mainly or exclusively male fruit flies, but the catches with EGOlure were equal or superior to those with TML. It is concluded that EGOlure should be considered as a suitable alternative for TML in detection, monitoring and control programs for African fruit flies of the genus Ceratitis. It has the added advantage that it combines the attractiveness with regard to species spectrum of both TML and terpinyl acetate.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.