S. Jayasankar

Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening

Seven ERF cDNAs were cloned from two Japanese plum (Prunus salicina L.) cultivars, ‘Early Golden’ (EG) and ‘Shiro’ (SH). Based on the sequence characterization, these Ps-ERFs could be classified into three of the four known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Functional nuclear localization signal analyses of two Ps-ERF proteins (Ps-ERF1a and -1b) were carried out using confocal microscopy. Expression analyses of Ps-ERF mRNAs were studied in the two plum cultivars in order to determine the role of this gene family in fruit development and ripening. The seven Ps-ERFs displayed differential expression pattern and levels throughout the various stages of flower and fruit development. The diversity in Ps-ERFs accumulation was largely due to the differences in their responses to the levels of ethylene production. However, other plant hormones such as cytokinin and auxin, which accumulate strongly throughout the various developmental stages, also influence the Ps-ERFs expression. The effect of the plant hormones, gibberellin, cytokinin, auxin, and ethylene in regulating the different Ps-ERF transcripts was investigated. A model was proposed in which the role played by the plant hormone auxin is as important as that of ethylene in initiating and determining the date and rate of ripening in Japanese plums.

Prunus domestica Pathogenesis-Related Protein-5 Activates the Defense Response Pathway and Enhances the Resistance to Fungal Infection

Pathogenesis-related protein-5 (PR-5) has been implicated in plant disease resistance and its antifungal activity has been demonstrated in some fruit species. However, their roles, especially their interactions with the other defense responses in plant cells, are still not fully understood. In this study, we have cloned and characterized a new PR-5 cDNA named PdPR5-1 from the European plum (Prunus domestica). Expression of PdPR5-1 was studied in different cultivars varying in resistance to the brown rot disease caused by the necrotrophic fungus Monilinia fructicola. In addition transgenic Arabidopsis, ectopically expressing PdPR5-1 was used to study its role in other plant defense responses after fungal infection. We show that the resistant cultivars exhibited much higher levels of transcripts than the susceptible cultivars during fruit ripening. However, significant rise in the transcript levels after infection with M. fructicola was observed in the susceptible cultivars too. Transgenic Arabidopsis plants exhibited more resistance to Alternaria brassicicola. Further, there was a significant increase in the transcripts of genes involved in the phenylpropanoid biosynthesis pathway such as phenylalanine ammonia-lyase (PAL) and phytoalexin (camalexin) pathway leading to an increase in camalexin content after fungal infection. Our results show that PdPR5-1 gene, in addition to its anti-fungal properties, has a possible role in activating other defense pathways, including phytoalexin production.

Differential regulation of four members of the ACC synthase gene family in plum

The regulation of ACC synthase (ACS) genes was studied in early (‘Early Golden’) and late (‘Shiro’) Japanese plum cultivars (Prunus salicina L.) in order to determine the role of this gene family in fruit ripening. Of the four Ps-ACS cDNAs isolated, two (Ps-ACS1 and -3) showed differential expression between the two cultivars. Ps-ACS1 accumulated during fruit ripening of ‘Early Golden’ (‘EG’) and ‘Shiro’ (‘SH’) in ethylene-dependent and -independent manners, respectively. Ps-ACS3a transcripts accumulated throughout fruit development and during ‘EG’ fruit ripening. Ps-ACS3b was detected only during ripening of ‘SH’ fruit. Furthermore, Ps-ACS3a transcript accumulation was negatively regulated by ethylene, whereas Ps-ACS3b was positively induced by the hormone. In both cultivars, the expression of Ps-ACS4 and -5 is under positive and negative feedback control by ethylene, respectively. Genetic analyses of ‘EG’ and ‘SH’ cultivars demonstrated that ‘EG’ is homozygous for Ps-ACS3a whereas ‘SH’ is heterozygous for Ps-ACS3 (a/b). The role of ethylene-overproducer 1-like in delaying fruit ripening by interacting with Ps-ACS proteins was also studied. The effect of the plant hormones, auxin, gibberellin, and cytokinin, in regulating ethylene production by promoting the induction of the different Ps-ACS mRNAs in plum was investigated. A model is presented in which differences in Ps-ACS alleles and gene expression between early and late plums are critical in determining the ripening behaviour of the cultivars.

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.

Regulation of two germin-like protein genes during plum fruit development

Germin-like proteins (GLPs) have several proposed roles in plant development and defence. Two novel genes (Ps-GLP1 and 2) encoding germin-like protein were isolated from plum (Prunus salicina). Their regulation was studied throughout fruit development and during ripening of early and late cultivars. These two genes exhibited similar expression patterns throughout the various stages of fruit development excluding two important stages, pit hardening (S2) and fruit ripening (S4). During fruit development until the ripening phase, the accumulation of both Ps-GLPs is related to the evolution of auxin. However, during the S2 stage only Ps-GLP1 is induced and this could putatively be in a H2O2-dependent manner. On the other hand, the diversity in the Ps-GLPs accumulation profile during the ripening process seems to be putatively due to the variability of endogenous auxin levels among the two plum cultivars, which consequently change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating Ps-GLPs transcripts was also investigated. These data, supported by their localization in the extracellular matrix, suggest that auxin is somehow involved in the regulation of both transcripts throughout fruit development and ripening.

Somatic embryogenesis in mango (Mangifera indica L.)

The mango Mangifera indica L. is considered to be one of the most important fruit crops of the world (Fig. 1). Its annual production is exceeded only by Musa (bananas and plantains), citrus, grapes and apples (FAO Production Yearbook, 1990). Currently, it is the most important fruit crop of Asia. Mango is in the Anacardiaceae family, which includes several other tropical and subtropical tree species of economic importance, e.g., cashew Anacardium occidentale, pistachio Pistachia vera, and several Spondias species, including S. cytherea, S. mombin and S. purpurea. The center of diversity for the genus Mangifera is in southeast Asia, with the greatest number of species being found on the island of Borneo. According to Mukherjee (1985), there are 39 currently recognized Mangifera species. The mango is the most widely cultivated species within the genus, and has a natural distribution throughout outheast Asia. It is found in forests as far west as the Indo-Burman region (Assam). There appear to be two distinct geographical races of the species: a polyembryonic race that occurs in the tropical rainforest of southeast Asia and a monoembryonic race that is associated with India. Some taxonomists have suggested that the mango originated in India, because of the ancient association between Indian culture and religion with this fruit. It has also been noted that the word for mango used throughout Asia is derived from the Tamil “maanga”.

Constitutive expression of Vitis vinifera thaumatin-like protein after in vitro selection and its role in anthracnose resistance

Anthracnose-resistant grapevine (Vitis vinifera L. cv. Chardonnay) plants were regenerated from embryogenic cultures that had been subjected to in vitro selection with culture filtrate of Elsinoe ampelina (de Bary) Shear. Three secreted proteins differentially expressed by in vitro-selected embryogenic cultures and regenerated plants were identified. An 8-kDa protein was identified as a lipid-transfer protein (LTP) by N-terminal amino acid sequence comparison. Two other differentially expressed proteins, with estimated molecular weights of 21.6 and 22kDa, immuno-reacted with antiserum raised against a thaumatin-like protein (TLP) protein from pinto bean (Phaseolus vulgaris L.). N-terminal amino acid sequencing of the 21.6-kDa protein showed a high degree of homology to V. vinifera thaumatin-like protein 2 (VVTL-2 = grapevine osmotin; Acc no. CAA71883), and that of the 22-kDa protein was homologous to V. vinifera thaumatin-like protein 1 (VVTL-1; AAB61590). Interestingly, both VVTL-1 and VVTL-2 are pathogenesis-related (PR) proteins, belonging to the PR-5 group. Protein produced from the cloned grapevine VVTL-1 gene significantly inhibited E. ampelina spore germination and hyphal growth in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in vivo. Enhanced expression of antifungal VVTL-1 in anthracnose resistant grapevine strongly suggests that it plays an important role, either alone or in conjunction with other PR proteins, by suppressing pathogen growth.

Characterization of embryogenic mango cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin

Abstract Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls.

Expression analysis of a plum pathogenesis related 10 (PR10) protein during brown rot infection

Plant PR10 is one of the pathogenesis related proteins, induced upon exposure to different stress conditions including fungal infection. PR10 proteins have been implicated in fungal disease resistance in some species; however its transcriptional regulation is not well understood. In the present work we cloned a PR10 gene from European plums (Prunus domestica L.) and monitored the quantitative changes in its transcript levels as a result of fungal infection in two varieties. We also studied the possible involvement of the membrane degrading enzyme phospholipase D-alpha (PLDα). In the susceptible variety, ‘Veeblue’, infection with the brown rot fungus Monilinia fructicola induced PLDα and PR10 expression, while in the resistant variety, ‘Violette’, a constitutive expression of PLDα and PR10 transcripts levels were observed. Resistance to M. fructicola also coincides with a sharp decrease in the expression of ABI1, a protein phosphatase and elevated hydrogen peroxide content after infection. Further, inhibition of PLDα by hexanal treatment, up-regulated ABI1 and decreased PR10 expression, suggesting a possible relationship between the two. We further confirm these results in Arabidopsis abi1 mutant that shows a higher level of PR10 transcripts.

Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum

Several dwarf plum genotypes (Prunus salicina L.), due to deficiency of unknown gibberellin (GA) signalling, were identified. A cDNA encoding GA 2-oxidase (PslGA2ox), the major gibberellin catabolic enzyme in plants, was cloned and used to screen the GA-deficient hybrids. This resulted in the identification of a dwarf plum hybrid, designated as DGO24, that exhibits a markedly elevated PslGA2ox signal. Grafting ‘Early Golden’ (EG), a commercial plum cultivar, on DGO24 (EG/D) enhanced PslGA2ox accumulation in the scion part and generated trees of compact stature. Assessment of active GAs in such trees revealed that DGO24 and EG/D accumulated relatively much lower quantities of main bioactive GAs (GA1 and GA4) than control trees (EG/M). Moreover, the physiological function of PslGA2ox was studied by determining the molecular and developmental consequences due to ectopic expression in Arabidopsis. Among several lines, two groups of homozygous transgenics that exhibited contrasting phenotypes were identified. Group-1 displayed a dwarf growth pattern typical of mutants with a GA deficiency including smaller leaves, shorter stems, and delay in the development of reproductive events. In contrast, Group-2 exhibited a ‘GA overdose’ phenotype as all the plants showed elongated growth, a typical response to GA application, even under limited GA conditions, potentially due to co-suppression of closely related Arabidopsis homologous. The studies reveal the possibility of utilizing PslGA2ox as a marker for developing size-controlling rootstocks in Prunus.