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.