Model plants used to better understand plant defense gene response to P. cinnamomi infection include; Zea mays, Arabidopsis thaliana, Lupinus angustifolius,Castanea sativa , Eucalyptus nitens, Lomandra longifolia, and most recently N. benthamiana . The gene expression in susceptible model hosts such as L. angustifolius and N. benthamiana can be compared to tolerant hosts like A. thaliana and L. longifolia to identify differences that may be associated with resistance to P. cinnamomi. Santos et al compared the gene expression between a susceptible and resistant variety of chestnut. They found that genes encoding for proteins involved in pathogen recognition proteins , were significantly upregulated in the resistant variety especially before inoculation. Six out of eight defense related genes including; WRKY31 and LRR-RLK’s were more highly expressed in the uninoculated C. crenata when compared to the uninoculated C. sativa. This increased basal defense to P. cinnamomi may contribute to this variety’s resistance. Gene expression in E. nitens in response to P. cinnamomi infection included up-regulated over represented gene ontology terms related to JA and ET signaling . Interestingly, pathogenesis-related gene 9 was down-regulated and represents a cross-species effector target during P. cinnamomi infection. Functional genomics and validation of these defense genes has only been performed in one study in A. thaliana. Eshraghi et al. reported that an auxin Arabidopsis mutant was more susceptible to P. cinnamomi infection than the wild type indicating the role of auxin pathways in P. cinnamomi defenses.The main challenges for the identification of P. cinnamomi resistance genes in avocado are the lack of tools available for functional genomic studies and limitations associated with tree crop biology.
Next-generation sequencing has provided some information on the expression of defense-related genes in avocado infected with P. cinnamomi. However, 25 liter plant pot the lack of the genome sequence and absence of functional genomic tools for avocado makes it difficult to determine and confirm their contributions to resistance against P. cinnamomi. The N. benthamiana model plant provides the opportunity to conduct functional genomic studies to determine the role of defense response genes to P. cinnamomi resistance that is not yet available in the avocado system or other tree hosts. Model plants including A. thaliana , L. angustifolius , and Medicago truncatula have been previously reported as susceptible hosts for this oomycete pathogen and have been used to study P. cinnamomi pathogenesis and plant responses to this pathogen. Although whole genome sequencing was available for these pathosystems, functional assays were not conducted with the exception of one study in Arabidopsis implicating the auxin signaling pathway with defense response against P. cinnamomi . Conducting RNAseq studies in N. benthamiana system at different time points during the infection process will provide a foot-hold into the defense gene expression pattern during P. cinnamomi infection and will allow us to conduct functional studies of selected defense genes using this N. benthamiana–P. cinnamomi pathosystem. Differentially expressed pathways and genes can be then validated by RT-qPCR in N.benthamiana and in avocado inoculated with P. cinnamomi using a detached leaf assay. Functional validation of the most promising genes can be done in N. benthamiana by transient over expression or silencing to determine their contribution to P. cinnamomi resistance. If similar expression patterns are found in avocado it is reasonable to consider this gene a good candidate for marker assisted breeding or biotechnology in avocado.
As genomic tools for avocado quickly become more available the methods developed in this system will become more applicable to this fruit tree crop. RNAseq analysis of infected N. benthamiana roots can complement this system by identifying what genes are universally expressed in the plant in response to P. cinnamomi infection and what gene expression is unique to the roots. Functional genomics are lacking in avocado; therefore, the objectives of this study were i) to establish a model system to look at defense gene expression in response to P. cinnamomi infection, ii) validate differentially expressed defense genes using overexpression in the same N. benthamiana model system, and iii) establishing connections to similarly expressed defense genes in avocado in response to P. cinnamomi infection. This information will help to select candidate defense genes in avocado for marker assisted breeding or biotechnology.Carbohydrates, mainly sugars and starch, are the major reserve, flavor, and textural components of many horticultural crops, and as such, determine their nutritional value, postharvest quality, and storage life. However, even in seemingly ‘carbohydrate-irrelevant’ leafy greens and various produce, starch and sugars may exert less obvious, yet we argue, critical roles in shaping post harvest quality. Starch and sugars have diverse functions in cells depending on their relative concentration, mobilization, subcellular location, and interaction with proteins. They sustain growth and buffer cells from stress, and as signaling molecules, they regulate many pathways that determine nutrient allocation to the sinks, and their partitioning into different biomolecular pools. Here, we intend to build several theoretical frameworks to show that carbohydrates, especially starch, may have ‘surprising’ roles in determining horticultural postharvest quality. We show that starch may be essential to, and intertwined with, climacteric ripening of fruits; starch is a determinant of leafy-green shelf life; sugars can influence the synthesis of specialized ‘sensory’ compounds; carbohydrates have roles in biotic and abiotic stress response and in determining fruit size; and that source tissue can determine sink quality.
We then point to molecular targets that can alter the carbohydrate profile of produce to obtain desirable traits.Starch and sugars accumulate in many fruits, tubers, and leaves, but with different temporal patterns and consequences for postharvest shelf life and quality . The timeframe over which the flux between starch and sugars occurs determines the classification and the role of carbohydrates. Harvested produce is often stored in the dark and at low temperatures, where respiration of reserves sustains the hexose phosphate pool . When reserves are exhausted, carbon starvation triggers senescence that manifests as spoilage. In tissues that accumulate starch as a carbon and energy reserve , granule degradation to sugar is surprisingly complex and multilayered. First, starch degradation occurs simultaneously with its biosynthesis at the granule surface even during the phase of net starch accumulation. This permits a bidirectional flow of carbon from starch to sugars, and potentially, to other compounds viathe hexose phosphate pool . Second, in fruit, in addition to exocorrosion at the surface, there is evidence of some endocorrosion in the starch granule, during ripening and fruit cold storage. Understanding the bidirectionality of flux between starch degradation and synthesis, and the physical organization of the starch granule associated enzymes, may offer opportunities to adjust reserve utilization during postharvest storage .The limited carbohydrate levels in leafy- and microgreens influence postharvest longevity by buffering against senescence. Shelf life is extended when harvested leaf starch levels are high, such as at the end of the day or after an extended light period. Starch content also positively correlates with desirable attributes such as sugar content, fresh weight, and texture. Identifying mechanisms that potentially coordinate photosynthesis, carbohydrate content, respiration, texture, and postharvest longevity in leafy greens is needed.Among fruits, the accumulation of high levels of starch appears to be a unique feature of those with climacteric ripening. In tomato fruit, starch may provide ∼40% of the carbon needed for fruit respiration. Furthermore, some of the fruit’s internal CO2 from respiration is likely fixed by fruit chloroplasts, contributing to ∼10–15% of ripe fruit carbohydrates. Transitory-storage starch may represent an evolutionary strategy for reproductive fitness with unintended benefits for the postharvest industry. First, its biosynthesis in climacteric species likely amplifies fruit sink strength, to undergird sink establishment and productivity. A large difference in sucrose concentration between source and sink, black plastic plant pots which occurs when imported sugars are converted to starch, would enable higher carbon allocation to fruit. Further, carbon storage as starch rather than as sugars minimizes cells’ osmotic disturbance. Second, increased fruit starch biosynthesis may also enhance plant survival under stress . From a postharvest perspective, the starch in climacteric-ripening fruit may be a vital energy source for maintaining biological processes, and for the synthesis of ‘quality-related’ metabolites that would minimize loss and waste . In contrast to the fruit described above, ‘sugar-storers’ are mainly non-climacteric and accumulate comparatively little starch . Furthermore, the starch is deposited in the peripheral regions of the fruit, and unlike the climacteric fruit, its accumulation peaks and is degraded to sugars, early in fruit development. However, starch in ‘sugar-storers’ may still contribute to fruit growth and quality by enabling a higher import of sugars into the developing fruit.Starch content and composition directly determine the functionality of starch in staple roots and tubers, but presumably, can also influence the biological processes of fruits and vegetables, which do not accumulate high levels of starch, as shown below: Produce firmness The crystalline and insoluble nature of starch directly contributes to the firmness of fruit and the texture of leafy vegetables.
When the dense granule is degraded to soluble sugars, intercellular space increases, thus promoting tissue softening. Tuber nutritional quality and textural attributes The relative proportion of the amylose and amylopectin fraction of starch is critical to tuber nutritional status and textural properties. Amylose is resistant to digestion and simulates fiber in the intestinal tract. Since amylose improves the nutritional value of starch-rich commodities, there have been many biotechnological efforts to increase its proportion relative to amylopectin in crops. Furthermore, the molecular structure of amylose is such that if high-amylose potatoes, cassava, and so on, are fried, they should have a crisper texture that may be desirable to consumers. In contrast, amylopectin provides smooth and moist textures to cooked starches which may be suitable for other end-uses. Sugar availability in fruit Starch granule crystallinity, composition, morphology, and size collectively influence starch degradation to sugars, which in developing fruit could have consequences for fruit respiration, metabolism, and ripening. We propose that the digestion of starch to sugars may be an inflection point for the rate of reserve use during ripening. Engineering starch with an optimal composition and crystalline structure to control the rate of release of sugars, may be valuable in regulating fruit metabolism, and hence quality.The relative sweetness of sugars varies as such: fructose > sucrose > glucose > sorbitol, so if the sugar content is the same, different proportions of sugars will give the fruit distinct taste and flavor profiles which are prime concerns for consumers. Modulating fructokinase activity and the SlSWEET sugar transporters in tomato increased fructose content, silencing A6PR reduced sorbitol relative to glucose in apple, and modifying PuWRKY31, promoted sucrose accumulation in pear. These changes should increase fruit sweetness, and, along with organic acids and aroma volatile compounds, should favorably influence fruit taste, flavor, and consumer likability.Sucrose and hexose have differential effects on fruit size, a trait determining consumer acceptance, shelf life, and nutritional characteristics. A high hexose-to-sucrose ratio in early fruit development stimulates mitotic activity that increases cell number through hexokinase signaling. Additionally, the higher osmotic potential of hexoses relative to sucrose will attract more water, increasing cell volume. Genes influencing fruit size, mediated in part by changes in carbohydrates, include the SWEET phloem sugar transporters in tomato and cucumber, the CsSUS4 gene in cucumber , and the SlCDF4 transcription factor and its Arabidopsis homolog in tomato. In apples, the sugar-to-acid ratio correlates with fruit size, which may have been selected through domestication. Although the mechanisms underlying these phenomena may vary, they show that carbohydrates are key determinants of fruit/organ size.Sugars both fuel and regulate the accumulation of specialized metabolites that are important to postharvest quality. Switches in the flux of carbon between primary and specialized metabolism were seen when sucrose was added exogenously to strawberry fruit, which inhibited the expression of many carbohydrate genes, but stimulated the MYB5 TF that regulates anthocyanin levels. Changes in flux were also seen when ectopic expression of the AtMYB12 TF in tomato reprogrammed carbon away from primary metabolism and toward flavonoid biosynthesis via the shikimate and phenylalanine pathways. There is a clear interrelation between primary and specialized metabolic pathways, but it remains relatively under investigated in horticultural crops. Identifying and modulating the TFs that regulate the fluxes between these pathways would enable the design of plants with a desirable combination of primary and secondary metabolites.Changes in plant carbohydrates, including the starch-tosugar conversion, are an important acclimatory response to stress, often with consequences for produce quality. For example, sugars maintain cells’ osmotic potential, provide energy for stress defense, and act as membrane protectants and ROS scavengers, as shown below.