Researchers from Cornell University and the U.S. Department of Energy's Brookhaven National Laboratory have unveiled a surprising function of a transport protein and its involvement in plant regulatory processes, offering potential benefits for addressing human mineral deficiencies. Published in The Plant Cell earlier this year, their study explores how understanding this protein could enhance the nutritional content of edible plant parts.
Iron, crucial for human health, serves as a vital component of hemoglobin, supporting oxygen transport, immune function, and cognitive processes. Since the human body cannot produce iron, regular consumption is essential. While plants like spinach are iron sources, strict regulatory mechanisms prevent excessive mineral accumulation, as high concentrations can be toxic to plants. Scientists have sought ways to override these regulations and boost the nutritional value of edible plants.
Olena Vatamaniuk, a plant biologist from Cornell leading the research, explained that the investigation began almost a decade ago when they discovered that the transport protein, oligopeptide transporter 3 (OPT3), moved iron within the model plant Arabidopsis thaliana, contrary to its name. Previous research from the University of Missouri had shown that reducing OPT3 affected iron distribution in A. thaliana, leading to signs of iron deficiency in the roots despite abundant iron in the leaves.
Building on these findings, Vatamaniuk and her team delved deeper into OPT3's role in shoot-to-root signaling. Utilizing ultrabright X-rays to examine the plants, they encountered yet another surprise from OPT3.
Shining a light on plant chemistry
When scientists seek to understand a protein's function, they often observe the consequences of its removal from a sample. Since completely removing OPT3 protein would be lethal to the studied plant species, researchers genetically altered the plants, creating mutants with reduced OPT3 abundance.
The focus shifted to comparing iron distribution in the vascular system between mutants and unaltered plants, particularly in the phloem—a transport tissue crucial for nutrient movement. Confocal X-ray fluorescence imaging (C-XRF), a technique developed by Cornell beamline scientist Arthur Woll, offered a way to analyze iron distributions at an ultra-small scale. This technique, employed at the National Synchrotron Light Source II (NSLS-II), provided single-micron resolution C-XRF images, an essential requirement.
At NSLS-II's Submicron Resolution X-ray Spectroscopy (SRX) beamline, led by Andrew Kiss, the researchers achieved an unprecedented level of detail. The X-ray beam, focused down to a single square micron on a plant's petiole, emitted fluorescent X-ray signals. These signals, collected by a nanofabricated confocal optic, underwent precise decoding to identify elements, concentrations, and locations within the sample.
Ju-Chen Chia, lead author and a researcher in Olena Vatamaniuk's lab, explained the original hypothesis about OPT3's role in loading iron into the phloem. However, their C-XRF analysis of mutant plant vascular tissues revealed a surprising outcome: more iron in the xylem but less in the phloem of the mutants, aligning with expectations. The unexpected insights derived from meticulous technical work underscore the complexity of plant regulatory mechanisms.
An unexpected finding
Examining transport proteins that handle multiple molecules is common in X-ray fluorescence experiments, especially in plants where minerals like iron are often transported alongside zinc or manganese. Therefore, researchers routinely analyze the distributions of various minerals simultaneously.
Ju-Chen Chia highlighted the importance of studying multiple essential minerals like iron, copper, zinc, and manganese concurrently, as changes in the concentration of one may affect others in plants. While copper typically has separate transporters, the unexpected discovery during their experiments at NSLS-II revealed that OPT3, responsible for iron transport, also moved copper into the phloem.
The revelation of OPT3 transporting both iron and copper was particularly surprising, challenging prior assumptions. Olena Vatamaniuk emphasized the significance of bringing their samples to NSLS-II for these unexpected insights, noting the unusual nature of one transporter handling multiple minerals in a plant.
Andrew Kiss acknowledged the technical achievement of the SRX beamline in facilitating this groundbreaking work. The collaboration and expertise at NSLS-II, involving Ryan Tappero from the X-ray Fluorescence Microscopy (XFM) beamline, played a crucial role in confirming and complementing the findings of Chia and her colleagues.
X-ray vision
At the XFM beamline, Cornell scientists aimed to visualize the elemental distribution within the vasculature of embryonic plants, contained within mature seeds. Unlike cutting open the seeds, which could cause element redistribution or chemical reactions, they opted for a non-invasive approach. Similar to medical CT scans, they used X-rays to perform a “chemical” CT scan of the mineral elements inside the seeds without physically opening them.
Ryan Tappero explained the process, likening it to medical CT scans but with a rotating seed instead of a rotating X-ray source. The half-millimeter-diameter seeds were ideal for intact scanning. Ultrabright X-rays were directed at the seeds, and as they rotated in the beam, fluorescence signals were recorded by a silicon drift detector. This X-ray fluorescence computed microtomography (F-CMT) technique provided cross-sectional images of the internal structures.
The F-CMT cross-sectional images, derived from fluorescence signals, offered insights into the internal distribution of elements in the embryonic plants. Observing lower concentrations of both iron and copper in the vascular cells of mutant seeds compared to unaltered seeds further supported the conclusion that OPT3 transported both minerals.
Ju-Chen Chia emphasized the significance of bringing samples to NSLS-II for a comprehensive understanding of the transport protein's physiology, noting that their work was nearing a pivotal moment. The combination of techniques and findings was bringing clarity to the intricate puzzle at the heart of their research.
Another chapter in the OPT3 story
Upon returning to their Cornell labs, the researchers delved into the genetic makeup of the mutant plants, unraveling a profound connection between iron and copper. Beyond sharing a transport protein, the two minerals engage in a complex signaling pathway regulating their uptake through gene expression—a pivotal discovery that advances efforts to modify nutrient content in edible plants, potentially addressing human mineral deficiencies.
Olena Vatamaniuk and her team focused on Arabidopsis thaliana, a non-grass plant renowned for its rapid reproduction and fully mapped genome. The researchers, armed with their newfound insights, now plan to extend their investigation to grass plants like rice, wheat, or barley.
“The physiology of a plant can influence transporter function,” Vatamaniuk noted, emphasizing the importance of applying this knowledge to diverse plant species. She anticipates more revelations as they expand their research.
Expressing gratitude to NSLS-II scientists, Vatamaniuk highlighted the significance of collaboration, praising the friendly and helpful nature of their counterparts. Ju-Chen Chia echoed the sentiment, acknowledging the vital role NSLS-II played in bringing their ambitious ideas to life.
Source: Brookhaven National Laboratory