Factors affecting grain set under drought are of special interest in commercial breeding endeavors.24
Because maize is an outcrossing species, pollen must move from the anthers at the top of the plant to the exposed silk of the same and surrounding plants. This growth stage in Croatia takes place at the beginning of July in mostly dry and warm weather conditions. Here, three maize hybrids were grown at two locations in eastern Croatia in two successive years (2008 and 2009). As shown in , the mean air temperature over the vegetation period of April to September was higher than the multiannual mean at both experimental localities, with 2009 being the warmest. 2009 was much dryer as well at both localities with 35% and 27% (Bicko Selo and Porec, respectively) less precipitation in comparison with multiannual mean. The effect of year was significant for leaf proline content () but unexpectedly it was significantly lower in 2009, whereas pollen proline content was higher in that year. Concurrently, the year effect was seen with both enzyme activities measured. P5CS-t activity was lower whilst PDH-t activity was much higher in 2009 when it was dryer and warmer. A possible explanation could be that inhibition of P5CS took place in such conditions. The very significant negative correlation between PRO-l and PDH-t (r = −0,937; p ≤ 0.01) indicates the possible utilization of proline for some purpose other than osmoprotection in 2009. As shown in , high TGN/e was accompanied with high PRO-l and low PDH-t. If proline content in the leaves was depleted for some reason, plant productivity declined. A possible reason for low proline accumulation in the leaves could be enhanced protein synthesis or proline translocation to other parts of the plant, such as generative organs, or the roots which have to grow into deeper soil levels under drought conditions. That suggestion is in concordance with the significantly higher proline content detected in pollen in 2009 and also with the results of Valentovič et al.25
who found that proline levels increased significantly under stress conditions in all studied tissues of 13-days old maize seedling. It was much higher in roots and mesocotyl tissues than in leaves of a drought-sensitive cultivar. Similarly, Raymond and Smirnoff26
stated that proline accumulation in maize root tips was the result of decreased oxidation and incorporation into protein at low water potential.
Climate characteristics-mean air temperatures and precipitation amounts at localities Bicko Selo and Poreč in experimental years 2008, 2009 in comparison with multiannual mean
Schafleitner et al.27
reported that the expression of both P5CS and PDH did not correlate with the proline levels found in leaf tissue indicating that mechanisms other than transcription participate in the regulation of proline accumulation in potato leaves. Chandra and Dubey28
reported that although the levels of proline increased with the magnitude of water stress the P5CS activity did not show a corresponding increase in all Cenchrus species which were examined. Here, the specific activities of P5CS and PDH showed a significant positive correlation (r = 0.942; p ≤ 0.01). This correlation along with an influence of the year in which they were grown was seen as both P5CS-s and PDH-s were lower in 2009, which was the dryer year. However there was also a higher total protein content found in leaf samples taken in 2009 (data not shown). It can be assumed that drought stimulated protein synthesis in the leaves, such as heat shock proteins, molecular chaperones, antioxidative enzymes or protein factors that might be involved in the regulation of signal transduction and expression of genes related to stress response, as reported by Marino et al.29
There was a large influence of both the locality in which they were grown and genotype on leaf, silk and pollen proline levels in the plants analysed. Plants grown at Porec had higher proline content in all tissues which were examined and both P5CS and PDH activities were lower in comparison with plants grown at Bicko Selo (). The estimated grain yield components were all influenced by year, locality and genotype (). Plant productivity was found to be higher in 2008 and at Porec, where higher proline levels in leaf, silk and pollen were observed. Taking into account that hybrid PR36B08 showed the lowest proline levels () but the highest DGM/p (), a significant relationship between proline content and metabolism on maize plant productivity are feasible, although this will be influenced by the agroecological conditions of the year and locality, as well as genotype of the plants grown. It is worth noting that the hybrid PR34F02 obviously had better pollination because it showed higher TGN/e, DGN/e, TGN/p and DGN/p than PR36B08, however the latters significantly smaller MDGN resulted in a lower DGM/p (). PR34F02 also had higher proline levels in leaf, silk and pollen, accompanied with less expressed enzymatic activity for P5CS and PDH (). This indicates that genotype effects on both proline metabolism and on grain set and development can not be neglected in maize plants.
Luna et al.30
reported that maize pollen dehiscence normally occurs in mid-morning when the temperature is typically increasing, relative humidity decreasing and radiation load increasing so that pollen loses viability quickly. Therefore, the prevention of water loss from both the pollen and stigma is very important for successful fertilization and seed set. In this context proline might be acting as an osmoprotectant at pollination. As mentioned above, hybrid PR34F02 had the highest silk and pollen proline level amongst the maize genotypes tested, and its grain productivity in terms of TGN/e and TGN/p was the highest. The main effect of heat stress during or after floral initiation is observed on kernel number.13
Grain numbers decrease because of several developmental changes, especially ovary abortion in maize or pollen sterility in small grains,12
as well as delayed silk emergence.15
Here, the abortion of grain during post-anthesis development was only 3% in PR34F02, 4% in PR36B08 and 6% in PR35P12, respectively.
In summary, here it was found that the measure of productivity TGN/e was positively correlated to proline content in the maize silk (PRO-s; ). Furthermore, multiple regression analyses showed a significant interplay between proline content (PRO-l, PRO-s), activities of enzymes involved in proline metabolism (P5CS-t, P5CS-s, PDH-t, PDH-s) and grain productivity of the plants (MDGM and DGM/p). However, it is quite obvious that proline content in maize tissues was influenced by various internal and external factors and single measurements can not reveal enough about the dynamics of proline metabolism and the roles of proline in plant growth, development, stress tolerance and productivity in field conditions. Hence, further evaluation in other environmental conditions, plant species and organs such as the root, would contribute to our knowledge on this exceptional amino acid and how it influences crop yields.