Short day promotes gall swelling by a CONSTANS-FLOWERING LOCUS T pathway in Zizania latifolia.

"Jiaobai" is a symbiont of Zizania latifolia and Ustilago esculenta, producing fleshy galls as a popular vegetable in South and East Asia. Current "Jiaobai" cultivars exhibit abundant variation in their gall formation date; however, the underlying mechanism is not clear. In this study, a strict short-day (SD) "Jiaobai" line "YD-3" was used. Plants were treated with two day-length regimes [14 h/10 h (day/night) (control) and 8 h/16 h (day/night) (SD)] from 100 to 130 days after planting. The gall swelling rate of the two treatments and another early SD treatment (from 60 to 90 days after planting), together with the contingent flowering plants in the experiment population, revealed that SD can improve both gall enlargement and flowering of "Jiaobai" plants. Comparison of RNA sequencing data among control, SD swelling, and SD flowering treatments of leaves and meristems indicated that SD promotion of "Jiaobai" swelling is conducted by the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway, similar but not identical to the SD-induced flowering pathway in Z latifolia and rice. "Virus-induced gene silencing", "Yeast one-hybrid assay" and "Dual-luciferase assay" showed that a FT gene, ZlGsd1, is critical in SD promotion of gall formation and is positively regulated by a CO gene, ZlCOL1. Our study elucidated how photoperiod affects the formation of a unique organ produced by plant-fungus symbiosis. The difference in SD response between "Jiaobai" and rice, as well as their potential applications in breeding of "Jiaobai" and rice, were also discussed.

The Roles of Hormone Signals Involved in Rhizosphere Pressure Response Induce Corm Expansion in Sagittaria trifolia.

Soil is the base for conventional plant growth. The rhizosphere pressure generated from soil compaction shows a dual effect on plant growth in agricultural production. Compacted soil leads to root growth stagnation and causes bending or thickening, thus affecting the growth of aboveground parts of plants. In arrowhead (Sagittaria trifolia L.), the corms derived from the expanded tips of underground stolons are its storage organ. We found that the formation of corms was significantly delayed under hydroponic conditions without rhizosphere pressure originating from soil/sand. In the initial stage of corm expansion, the anatomic structure of arrowhead corm-forming parts harvested from hydroponics and sand culture was observed, and we found that the corm expansion was derived from cell enlargement and starch accumulation. Comparative transcriptome analysis indicated that the corm expansion was closely related to the change in endogenous hormone levels. Endogenous abscisic acid and salicylic acid concentrations were significantly increased in sand-cultured corms. Higher ethylene and jasmonic acid contents were also detected in all arrowhead samples, demonstrating that these hormones may play potential roles in the rhizosphere pressure response and corm expansion. The expression of genes participating in hormone signaling could explain the rising accumulation of certain hormones. Our current results draw an extensive model to reveal the potential regulation mechanism of arrowhead corm expansion promoted by rhizosphere pressure, which will provide important references for further studying the molecular mechanism of rhizosphere pressure modulating the development of underground storage organs in other plants.

Selection of Reliable Reference Genes for Gene Expression Normalization in Sagittaria trifolia.

Real-time quantitative PCR (RT-qPCR) is a method with high sensitivity and convenience that has been extensively used to analyze the expression level of target genes. A reference gene with a highly stable expression is required to ensure the accuracy of experimental results. However, the report on appropriate reference genes in arrowheads (Sagittaria trifolia) is still limited. In this study, eight candidate reference genes (ACT5, UBQ, GAPDH, CYP, NAC, IDH, SLEEPER and PLA) were selected. The candidate genes were employed in a RT-qPCR assay in different tissues at different developmental stages of the same tissue (including corm, leaf and leafstalk) in arrowheads. Five statistical algorithms, GeNorm, NormFinder, BestKeeper, delta cycle threshold (ΔCt) and RefFinder, were used to evaluate the stability of these genes' expressions in order to identify the appropriate reference genes. The results showed that UBQ was the optimum reference gene in leaf, leafstalk, root, stolon and corm, IDH exhibited the most stable expression during the expansion of corm, UBQ and PLA were the most stable reference genes in developmental stages of leaf and leafstalk, respectively. Finally, the reliability of reference genes was further confirmed by the normalization of PDS and EXP1 genes under different arrowhead tissues and developmental stages of corm, respectively. This study constitutes important guidance for the selection of reliable reference genes for analyzing the tissue- and developmental-stage-specific expression of genes in arrowheads.

Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation.