The observations suggest that OsZHD2 increases the amount of local auxin occurring in the dividing zone of the roots. We propose that OsZHD2 induces auxin biosynthesis in the RAM by increasing ethylene levels. However, we do not rule out the possibility that OsZHD2 directly increases auxin levelsby controlling other genes that we did not investigate in the present study . OsZHD2 induces root development by increasing ethylene biosynthesis and sequentially auxin biosynthesis. It determines meristem-specific homeobox protein functions as an activator for meristem activity by regulating the ethylene–auxin interaction. In the RAM region, ethylene–auxin crosstalk plays important roles .Root system architecture is a critical agronomic trait that influences crop productivity by altering soil mineral absorption and lodging . Deep rooting is a key trait that facilitates drought stress tolerance, since plants can absorb water from deeper soil layers . In addition, the introgression of the DEEPER ROOTING 1 allele from a deep-rooting rice cultivar into a shallow-rooting rice cultivar increases yield under drought conditions , while root-specific over expression of OsNAC5 enhances root diameter, resulting in greater drought tolerance and higher grain yield . Here, we demonstrated that the over expression of OsZHD2 increases the volume of the root system and overall yield, particularly under a poor nutritional status .
Therefore, our results suggest that OsZHD2 is a key trait that could be applied in the improvement of grain yield.We observed that the increased expression of OsZHD2 stimulated root growth. The effect was more significant for lateral roots. Although the total number of lateral roots increased due to the over expression of the gene,plastic pots plants their density did not change . Therefore, the function of OsZHD2 seems to be associated primarily with root growth rather than root initiation. In situ RNA hybridization analyses revealed the preferential and uniform expression of OsZHD2 in the lateral root meristem region, supporting the root growth function . The number of dividing cells in the RAM region was significantly higher in the OsZHD2-OX plants, further indicating that the gene stimulates root growth . The process of initiating lateral roots has been elucidated extensively using numerous mutants defective in that step. However, the molecular mechanisms of lateral root emergence and growth remain poorly understood . Mutations of orc3 in rice interrupt the cell cycle process and block lateral root emergence . The ORC is a critical element in DNA replication, cell cycle checkpoint regulation, heterochromatin assembly, and chromosome assembly. The expression levels of the genes of the D-type cyclin family are down-regulated significantly in orc3 mutants . In the present study, the expression levels of CYCD4;1 increased in OsZHD2-D, suggesting that OsZHD2 promotes cell cycle progression during lateral root growth .
Plants rely on an array of phytohormones to coordinate and finely regulate response networks to biotic and abiotic stresses . Studies of phytohormone regulation of defense responses in plant–microbe interactions generally have focused on salicylic acid , jasmonic acid , and ethylene . In Arabidopsis, SA-mediated defenses are considered deterrents to biotrophic pathogens, whereas JA/ET-mediated defenses deter necrotrophic pathogens . However, this dichotomy with respect to parasitic strategy may be less clear in other host species . While studies of SA and JA/ET signaling have shaped our current understanding of induced resistance mechanisms, consideration of other phytohormones is gaining traction in terms of how multiple stress response pathways overlap as non-linear networks to coordinate plant responses to diverse biotic challengers . These interactions can be synergistic or antagonistic, or phenotypically neutral if the disease assays cannot discern subtle differences. For example, SA and JA can be antagonistic in certain contexts leading to trade-offs in defense against different attackers . Similarly, there is substantial evidence that elevated levels of the phytohormone, abscisic acid , can diminish host resistance . Nonetheless, ABA enhances resistance in some biotrophic and other interactions . This parasitic context dependency of ABA action illustrates the challenge in identifying a unifying mechanism to explain ABA’s effects in plant–microbe interactions.
Various root stresses reduce plant water potential and induce ABA accumulation to trigger adaptive biochemical and physiological changes that enable plants to maintain water balance . However, episodic root stresses, even those from which plants fully recover, occur routinely in agricultural and natural systems, transiently elevating cellular ABA concentrations to levels that predispose plants to inoculum densities they would normally resist . Disease predisposition from abiotic stress has long been recognized in the plant pathology literature , and is particularly well-documented in classic studies of root and crown diseases caused by Phytophthora spp., where episodes of water logging, soil salinity, and drought are important factors in disease development . Although a role for ABA in conditioning the increased susceptibility during and following stress episodes is recognized, the underlying mechanisms and impacts on host defenses are unresolved . Furthermore, relatively little attention has been directed at defense-related phytohormone signaling in root–pathogen interactions where the predisposing stresses of water deficit, hypoxia and soil salinity are encountered most directly . Salicylic acid is involved in multiple physiological processes , but is perhaps most studied for its role in systemic acquired resistance and as a strong inducer of pathogenesis-related proteins . SA biosynthesis in plants occurs by two pathways, one via isochorismate synthase , and the other via phenylalanine ammonia lyase . Knockout mutants in the ICS pathway and transgenic plants carrying nahG encoding a bacterial salicylate hydroxylase have reduced SA levels, are highly susceptible to pathogens, have severely reduced levels of PRproteins, and fail to develop local and systemic resistance . ABA appears to antagonize SA action in defense . Jasmonic acid is an oxylipin involved in defense responses against necrotrophic pathogens and insect herbivores, and has been found to have positive or negative interactions with SA depending on the specific host-parasite/pest context . JA also acts synergistically with the phytohormone ET, and either synergistically or antagonistically with ABA . In soybean leaves, JA levels increase with ABA during dehydration, and a few studies have shown ABA signaling necessary for JA biosynthesis and elaboration of defense responses . In Arabidopsis, application of ABA suppresses some JA/ET activated genes such as PDF1.2, while JA/ET responsive genes are up-regulated in ABA-deficient mutants such as aba1 and aba2 . Several JA synthesis mutants are available in tomato, including defenseless-1 , blocked in the conversion of 13-hydroperoxylinoleic acid to 12-oxophytodienoic acid, and acx1, a mutant defective in acyl-CoA oxidase . These mutations result in reduced JA accumulation and pathogen-related transcripts . Phytophthora capsici is a broad host-range pathogen that can cause significant economic losses in vegetable crops in the Cucurbitaceae, Solanaceae, and Leguminosae families . Similar to other soilborne Phytophthora species, P. capsici causes extensive root and crown rots that are exacerbated by predisposing stresses such as water logging and salinity. In a previous study, we imposed acute levels of salt stress on hydroponically grown tomato seedlings prior to inoculation with P. capsici to show that predisposition in roots and stems occurred in an ABA-dependent and ET-independent manner . In a related study ,plastic nursery pots we showed that plant activators that engage SA-mediated defenses in tomato induce resistance to the bacterial speck pathogen, Pseudomonas syringae pv. tomato , both in non-stressed and salt-stressed plants, but not in the case of P. capsici where plants exposed to these same treatment regimes displayed similar severity of root and crown rot.
The objective of this study was to further assess the impact of salt stress on the infection and colonization of tomato roots by P. capsici and to determine if there is discernible interaction between ABA and SA or JA during salinity-induced predisposition. We examined the impact of salt stress on P. capsici zoospore attraction and early infection and colonization in tomato roots in wild-type and ABAdeficient mutants. Since ABA can alter the action of SA and JA , we evaluated SA- and JAdeficient tomato plants for altered predisposition phenotypes. In addition, we profiled the expression of hallmark genes for stress adaptation and defense during predisposition onset and recovery and P. capsici infection. Tomato plants of cultivars ‘New Yorker,’ ‘Rheinlands Ruhm,’ or ‘Castlemart’ and mutant or transgenic lines within these backgrounds were used in experiments. ‘New Yorker’ and ‘Rheinlands Ruhm’ are determinate and indeterminate cultivars, respectively, used primarily for fresh market consumption, and ‘Castlemart’ is a determinate, processing cultivar that was bred for the arid growing conditions of California and other regions. In our experimental format, all three cultivars are susceptible to P. capsici. ‘New Yorker’ seeds were obtained from a commercial source . The homozygous ABA-deficient mutants sitiens and flacca were compared with their isogenic, wild-type background, ‘Rheinlands Ruhm,’ and seeds for these were obtained from the C. M. Rick Tomato Genetics Resource Center, University of California, Davis. ‘Rheinlands Ruhm,’ sitiens, and flacca plants were grown for seed production in the greenhouse. NahG transgenic plants were generated in the ‘New Yorker’ background, similar to the method used by Gaffney et al. . The nahG construct containing the transgene salicylate hydroxylase under control of the CaMV 35S promoter in the binary vector pCIB200 was a gift of Syngenta Crop Protection, Inc. SA deficiency of our transgenic line was confirmed previously . The acx1 and def1 mutants in the cv. ‘Castlemart’ background were a gift of Gregg Howe, Michigan State University. Seeds of ‘Castlemart’ were obtained from the C. M. Rick Tomato Genetics Resource Center. Four-week-old plants with two or three true leaves were grown hydroponically as described previously . Experiments were conducted in a growth chamber . A pepper isolate of P. capsiciwas used for most experiments. A P. capsici isolate transformed with the green fluorescent protein was a gift of Christine Smart and William Fry, Cornell University . Wildtype and transformant P. capsici strains were maintained on V8 juice agar plates or V8 juice amended with 100 mg/L geneticin , respectively. Zoospore inoculum was prepared using methods described previously .The salt stress regime selected for these experiments was based on prior studies of root stress predisposition . The impact of salinity stress differs from other osmotic dehydration stresses primarily in that salt-stressed plants are additionally exposed to abnormally high extracellular concentration of ions such as sodium and chloride . The inclusion of calcium helps to mitigate the confounding toxicity caused by sodium and emphasizes the osmotic facet of salinity stress, which is likely applicable to other dehydration stresses . Plants were subjected to salt stress by replacing the 0.5X Hoagland’s solution with 0.2 M NaCl and 0.02 M CaCl2 for 18 h. Plants were returned to 0.5X Hoagland’s solution, allowed to regain turgor and recover for 2 h, and then inoculated with zoospores of P. capsici . To determine whether there was an effect on zoospore motility and chemotaxis, a microcapillary swim-in assay similar to that described by Morris and Ward was used with exudates collected from tomato roots. Following 18 h salt stress, tomato roots of uniform volume were rinsed in deionized H2O and transferred to tubes containing 2 ml of deionized water. Exudates were allowed to accumulate for 2 h, tomatoes were then removed, and the exudates were vortexed and immediately loaded into 1 µl microcapillary tubes . Exudate-loaded microcapillaries were placed into 15 cm petri dishes with one end submerged in a 500 µl droplet of 5 × 105 zoospores ml−1 . Microcapillaries were photographed under a dissecting microscope 15 min after being placed into the zoospore suspension. Zoospore attraction was determined as the proportion of the microcapillary’s inside diameter blocked by encysted zoospores and scored on a 0–5 rating scale .The P. capsici-GFP transformant was visualized 24 and 48 hours post inoculation in tomato roots using a Leica TCS SPE confocal system . Following infection and just prior to microscopy, roots were dipped into a 10 µg/ml solution of propidium iodide for 30 s and rinsed in sterile water before mounting on microscope slides . GFP was excited at 488 nm and emission was collected between 510 and 550 nm. PI was excited at 534 nm and emission was collected between 600 and 650 nm. Laser power was set to 50% with a gain of 800–900 for both the 488 nm and 534 nm channels. Final images were composites of five Z steps through root tissues approximately 40 µm in depth.To estimate the progression of P. capsici colonization in tomato seedlings by qPCR, nahG, def1, and acx1 plants and wild-type plants of their corresponding backgrounds were frozen in liquid N2 at 48 hpi, and stored at −80◦C until extraction and analysis.