Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.)

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the in-vitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277–1289; N. Sinha et al., 1993 Genes Dev. 7: 787–797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro. Received: 2 June 1997 / Accepted: 21 July 1997     

Exogenous sucrose utilization and starch biosynthesis among sweetpotato cultivars

Three sweetpotato cultivars were investigated for their starch content and amylose/amylopectin ratio. Ym starch contains 87.2% amylopectin and 12.8% amylose, when total starch was calculated as 100%. The Zm cultivar contains 33.6% amylopectin and 18.2% amylose, and its total starch was calculated as 51.8% of that of Ym. The Hm cultivar contains 39.1% amylopectin and 30.5% amylose, and its total starch was 69.6%. We analyzed the expression levels of starch and sucrose biosynthesis-related genes including AGPases a, b, and c; sucrose synthases I and II; starch synthase I; GBSS I; and SBEs I and II. All genes tested in this experiment were detected only in Ym, while several genes showed very faint or no expression in Zm and Hm. We also measured tissue-specific expression of these genes in whole plants of Ym. Most of the genes are expressed in the stem and roots of the plants. Expression profiles of starch synthesis-related genes of the sweetpotato leaves were investigated after supplementing the different concentrations of sucrose solution. All genes in Ym were clearly induced by sucrose, but the expression levels of some of these genes did not change in Zm and Hm. The total starch content of Ym, Zm, and Hm gradually increased over time on addition of 3%, 6%, and 9% sucrose concentrations. The greatest accumulation was observed in Ym at 48 h, and it was almost 2.24 times higher than that of the (0%) control, while Zm and Hm showed 1.76 and 1.91 times higher levels of starch, respectively. These results indicate that cooperative expression of all related genes is essential for starch biosynthesis from sucrose. This is the first report on different sucrose contents and the efficiency with which exogenous sucrose switches on gene expression of starch biosynthesis-related genes among cultivars.     

Overexpression of <Emphasis Type="Italic">Zm401</Emphasis>, an mRNA-like RNA,has distinct effects on pollen development in maize

We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development, the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.