The occurrence of the sheathed panicle in rice is mostly related to the insufficient elongation of the longitudinal cells in stem [12]. TaKeda et al have classified the shortened stem types into five categories based on the proportional ratio of the length of each stem internodes to the whole plant height in rice [13]. Among them, the "sh" type was that the shortening occurs specifically at the uppermost internode. In this situation, the uppermost internode almost has no elongation, and the panicle was sheathed by the flag leaf completely. However, the "nl" type was that the elongation of the uppermost internode was shortened and the basal internode was elongated. According to the phenotype of sheathed spike caused by TaWUS-like, we defined the shortening type as "nl", which is characterized by an obvious shortening of the uppermost internode and a slightly elongation of the basal internode. Although the last internode could completely disappear in some TaWUS-like-OE lines (Fig. 1C), the length of the base internode was always longer than that of WT (P < 0.05), indicating the occurrence of sheathed spike in wheat may also be related to the insufficient elongation of stem cells. In rice, researchers have found kinds of shortening types of internodes [12, 15–17]. Wang et al obtained sui(t) mutant from JinHui 10 of the Indica restorer line using the method of EMS mutation showing that an extremely shortened uppermost internode with the rice panicle full-wrapped in the leaf sheath, while the other internodes were not changed [15]. Another specific gene esp2, mapped on rice chromosome 1, was also showed to control the development of rice uppermost internode. The uppermost internode of monogenic recessive mutant was almost completely degenerated and panicle was completely sheathed by the flag leaf, while the length of the other internodes did not change significantly [12]. In the following researches, it has proved that sui1-1, sui1-2 and esp2 actually were the allelic mutations for each other, encoding a phosphatidylserine synthetase, and the mutation type belong to "sh" type [16]. Meanwhile, the rice full-wrapped mutant fsp, the shortening was occurred from the first internode to the last internodes. However, the proportion of shortened internodes to mutant height is the same as those in WT, thus, mutation belongs to "dn" type [17]. Different genes in different crops caused different shortening phenotype of stem may indicate a complex and different network regulating the stem development. Therefore, we suggested that some mechanisms regulating internodes elongation may also be relatively conservative among different gramineous crops.
GA and BR are important two hormone regulators affecting crop architecture and yield [18]. It has been reported that the development of rice uppermost internodes is mainly regulated by GA and BR [5, 11, 19]. In this experiment, the GA and BR contents in flag leaves and internodes of wheat at heading stage were evaluated. Results showed that the GA3 content in the uppermost and secondary internodes of TaWUS-like-OE lines decreased about 2 times (P < 0.01) and 1.7 times (P < 0.05) compared with that of WT, respectively, while the BR content increased about 2.3 times (P < 0.01) and 2.2 times (P < 0.05), respectively. These results indicated that the dwarfing of TaWUS-like-OE lines and the shortening of internodes may be closely related to the decrease of GA content and the increase of BR level, which is consistent with the research in rice. The reasons for that phenotypes are that the genes regulating the changes of internodes are mostly related to GA synthesis [20, 21], catabolism[22] and signal transduction [11, 23]. Thus, this may verify that the mechanism of GA regulating internodes elongation could be a relatively conservative mechanism in different gramineous crops.
In rice, it has also been found that the elongation of internodes was regulated by BR [5, 24]. As reported by Sun et al, SUI2 encodes a cytochrome oxidase. The dominant mutation of this single gene can cause a significant shortening of the uppermost internode in rice, which is closely related to the effect of sui2 on the expression of BR signaling related genes and the insufficient elongation of the longitudinal cells in stem [5]. Yamamuro et al found a rice dwarf mutant d61 which is insensitive to BR. The BR level in mutant d61 was higher than that of the WT, meanwhile, the longitudinal elongation of cells was reduced, and the arrangement of microtubules was distorted [24]. In our study, we found the BR content in the internodes was increased, and the longitudinal elongation of stem cells in the pith region was significantly reduced in TaWUS-like-OE lines. In addition, the shapes of about 80% of the parenchyma cells in the pith region changed from a regular rectangle to an irregular shape, and the linear arrangement was distorted. Combined with previous studies and our results, we believed that BR was involved in the regulation of the formation and normal elongation of intercalary meristem, but we speculated that BR may affect the formation of internodes earlier than GA, so that the disorder of cell microtubule arrangement was observed in the stem tissues with obvious changes of BR content, while no changes was found for cell shape or arrangement in the stem tissues with only significant changes of GA. Therefore, we believe that the shortening of internodes of wheat is related to the obvious inhibition of cell division and longitudinal elongation by TaWUS-like, while GA and BR are together involved in the regulation of genes expression of stem development, which is different from the other reports that GA or BR just alone regulate internode elongation [5, 20, 25].
Changes in hormone levels are mostly related to the changes of genes expression involved in hormone synthesis or signal transduction. In order to learn the expression characterization of GA- and BR-related genes and understand the molecular mechanism of sheathed spike genes, we analyzed downstream regulatory genes of TaWUS-like. Results showed that the expression levels of key genes related to GA synthesis and signal transduction were significantly inhibited, while the activities of the inactivation-related genes were significantly increased. The gene activities of serine/threonine protein phosphatase related to the regulation of phosphorylation level in the BR signaling pathway were significantly inhibited by 2.1 times and 2.8 times respectively, while the activities of SMT, det and Dim, the key genes of BR synthesis pathway, were significantly inhibited and activated, respectively. The results were inconsistent with the view of Sun et al that the regulation of rice stem elongation by sui2 was unrelated to the genes involved in BR synthetic pathway. This indicated that the sheathed spike gene not only affected hormone content but also regulated hormone signal transduction in the stems. In addition, there should exists an unknown balance between GA and BR signals, the detailed mechanism needs more researches to explore.
WUSCHEL-related homeobox family genes play important and various roles in plant growth and development [26]. To date, just one WUSCHEL- related homeobox family gene, DWARF TILLER1 (DWT1) [27], was found to regulate the elongation of intercalary meristem of rice, which is homologous to the members of WOX8 and WOX9 in Arabidopsis thaliana and controls the developmental uniformity of the main shoot and tillers in rice. In dwt1 mutant, the 2nd, 3rd and 4th internodes were significantly shortened, but the elongation of the uppermost internode almost was not affected, which has a great difference with the phenotype of TaWUS-like-OE. In our TaWUS-like-OE lines, the shortening of internodes occurred specifically in the uppermost and secondary internode. Besides, two genes belong to different subfamily of WUSCHEL-related homeobox family based on the comparison of amino acid sequence. DWT1 belongs to intermediate type, while TaWUS-like belongs to WUSCHELE type, which is highly homologous to the wox5-like gene in rice and Aegilops chinensis, respectively. In our knowledge, this is the first important gene reported in wheat that have a function in regulating the elongation of the internode. Results indicated that different WUSCHEL family members may have different mechanisms in regulating stem development. In addition, both we and Wang et al. found that these two genes were highly expressed in young spikes and developing grains, but could not detected in the internodes (data not shown). Therefore, we suggest that the WUSCHEL family genes should have a key regulatory point or share a common mechanism, which may transfer an unknown signal or small molecular substances from the grains to the internodes in timely and correctly thus significantly affecting the elongation of the plant stems. In the study of rice, Wang et al believed that DWT1 may affect cell division and elongation via regulating the genes expression of cyclin and expansin. Among the changes of the expression of downstream genes regulated by TaWUS-like, we also found the activities of 8 cyclins and 8 expansin belong to EXPA/EXPB family were significantly inhibited. Wang et al has been reported that the cyclins and its dependent protein kinases together are closely related to cell division and differentiation, while the expansin of EXPA/EXPB family is highly related to cell wall loosening and cell elongation. Therefore, the decreased activities of these genes could directly lead to the retarded of cell division and insufficient elongation of longitudinal cells in stem.