Xylem functionality controlling blossom-end rot incidence in transgenic ALC::NCED tomato plants

Authors: RIBOLDI, LUCAS BAIOCHI - Universidad De Sao Paulo; DE FREITAS, SERGIO TONETTO - Brazilian Agricultural Research Corporation (EMBRAPA); Norris, Ayla; Jiang, Cai-Zhong

Submitted to: South African Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2022
Publication Date: 7/15/2022
Citation: Riboldi, L., de Freitas, S., Norris, A.M., Jiang, C. 2022. Xylem functionality controlling blossom-end rot incidence in transgenic ALC::NCED tomato plants. South African Journal of Botany. 150:120-128. https://doi.org/10.1016/j.sajb.2022.07.015.
DOI: https://doi.org/10.1016/j.sajb.2022.07.015

Interpretive Summary: Blossom-end rot (BER) is a physiological disorder characterized by a water-soaked tissue evolving into necrotic tissue, caused by a rupture in the cell wall and membranes in the distal part of fleshy fruits, like tomatoes, peppers, and watermelons. For many years, BER incidence has been known to be related to calcium (Ca2+) concentration and distribution in leaves and fruit, but recently, new studies proposed that other factor could also determine fruit susceptibility to BER, such as the number of functional xylems, Ca2+ uptake, and oxidative stress in the fruit. Water transport, nutrients, and other metabolites move through plants mainly via transpiratory flow from the roots to the aerial parts, driven by high-growth and intense transpiration zones in meristems, leaves, and fruit. Ca2+ translocation occurs exclusively via xylem and is determined by transpiration and growth rates of different plant organs. As Ca2+ transport is coordinated by transpiratory flow and carried to the fruit via xylem, it is necessary that its xylem vessels remain functional, mainly in the distal portion of the fruit, inhibiting BER development. During early stages of growth and development, fruit present a higher number of functional xylems, which decreases toward fruit maturation possibly due to the accumulation of substances, such as callose, leading to physical blockage, embolism, and loss of the hydrostatic gradient between the peduncle and distal ends of the fruit. The decrease in xylemic flow starts from minor veins in the distal portion of the fruit towards the major bundles at the proximal end, and at maturity, xylem transport is restricted to a small portion of tissue located between the receptacle at the pedicel end and the cavity just proximal to the pit, making it impossible for water and particularly Ca2+ to reach the distal portion of the fruit. One approach that has being used to increase and/or maintain higher xylem functionality during fruit growth and development is the application of abscisic acid (ABA). ABA is a well-studied plant hormone, and its concentration rapidly increases during stress conditions. ABA signaling induces stomata closure, decreasing plant water loss via transpiration, leading to an increased plant water use efficiency (WUE), which can also help mitigate the negative effects of drought and high temperatures on fruit production. Although exogenous ABA applications have been shown to increase plant resistance to abiotic stresses, an interesting alternative to external ABA application is the manipulation endogenous ABA concentration within plant tissues. ABA content can be manipulated by increasing the expression of genes coding for enzymes involved in ABA biosynthesis, mainly its rate-limiting enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). Previous studies had success controlling NCED expression using either a constitutive promoter or an inducible system, with subsequent increase of ABA, morphological and physiological changes. However, those approaches present some technical problems to study BER. First, constitutive promoters, like Cauliflower Mosaic virus 35S (CaMV 35S), directs gene expression uniformly in most tissues, cells at all stages of plant growth and development, and a varied expression effects result from its interaction with environmental factors and the physiological state of the plant’s development. Inducible promoters, triggered by physical or chemical factors, have been shown to be a powerful tool to regulate the expression of genes at certain stages of plant or tissue development. In Arabidopsis, an example is RD29 genes, in which two genes, RD29A and RD29B, are stress induced, where RD29A is induced by drought and cold and RD29B by salt stress. However, as discussed above, BER and NCED are also triggered by drought. Thus, the response promotion drought activated can impact in BER developm

Technical Abstract: Fruit susceptibility to the physiological disorder known as blossom-end rot (BER) is an important limitation in tomato production. Abscisic acid (ABA) is known to reduce leaf transpiration, which can enhance plant water use efficiency (WUE), as well as increase fruit xylem functionality, Ca2+ uptake and oxidative stress defenses, which has been suggested to reduce BER incidence. However, the role of ABA on most of these factors determining fruit susceptibility to BER remains poorly understood. ABA production is mainly regulated by the expression of 9-cis-epoxycarotenoid dioxygenase (NCED) genes coding for the key enzymes involved in ABA biosynthesis. Manipulation of NCED gene expression by the alcohol inducible promoter (ALC) could be an alternative approach to stimulate ABA production and its beneficial effects on inhibiting BER incidence. The objectives of this study were to use ALC::NCED transgenic tomato plants to manipulate NCED expression and ABA production in order to improve plant WUE, and increase fruit xylem functionality, Ca2+ uptake and oxidative stress responses, reducing losses due to BER incidence. In this study, two transgenic tomato lines (1 and 2) were developed with the ALC::NCED construct. This construct allows the inducible activation of the ALC promoter by treating the plants with ethanol vapor that drives NCED expression and ABA synthesis. According to the results, after full bloom, weekly spraying transgenic plants with ethanol (2%) decreased BER incidence, compared to the wild type ‘New Yorker’ plants. The transgenic line 1 had higher NCED expression in response to ethanol than the transgenic line 2 and wild type ‘New Yorker’. At 15 and 30 days after pollination, transgenic lines 1 and 2 had higher number of functional xylem vessels, as well as higher Ca2+ concentration in the distal end of the fruit, compared to the wild type fruit. WUE was higher in the transgenic lines than in the wild type. Both transgenic lines also showed higher antioxidant content in leaves and fruit. Therefore, our study shows that stimulating NCED expression with an inducible system increases the number of functional xylems and Ca2+ uptake into the fruit, improving plant WUE and reducing BER incidence.